Nano gold conjugation, anti-arthritic potential and toxicity studies of snake Naja Kaouthia (Lesson, 1831) venom protein toxin NKCT1 in male albino rats and mice

Indian Journal of Experimental Biology

Vol. 52, August 2014, pp. 763-772

Nano gold conjugation, anti-arthritic potential and toxicity studies of snake Naja

kaouthia (Lesson, 1831) venom protein toxin NKCT1 in male albino rats and mice

Partha Pratim Saha1, Tanmoy Bhowmik

1, Anjan Kumar Dasgupta

2 & Antony Gomes

1*

1Laboratory of Toxinology & Experimental Pharmacodynamics

Department of Physiology, University of Calcutta, 92 A P C Road, Kolkata 700 009, India 2Department of Biochemistry, University of Calcutta

35 Ballygunge Circular Road, Kolkata 700 019, India

Received 20 January 2014; revised 8 May 2014

Nanoscience and Nanotechnology have found their way in the fields of pharmacology and medicine. The conjugation

of drug to nanoparticles combines the properties of both. In this study, gold nanoparticle (GNP) was conjugated with

NKCT1, a cytotoxic protein toxin from Indian cobra venom for evaluation of anti-arthritic activity and toxicity in

experimental animal models. GNP conjugated NKCT1 (GNP-NKCT1) synthesized by NaBH4 reduction method was stable

at room temperature (25±2 °C), pH 7.2. Hydrodynamic size of GNP-NKCT1 was 68–122 nm. Arthritis was developed by

Freund's complete adjuvant induction in male albino rats and treatment was done with NKCT1/GNP-NKCT1/standard drug.

The paw/ankle swelling, urinary markers, serum markers and cytokines were changed significantly in arthritic control rats

which were restored after GNP-NKCT1 treatment. Acute toxicity study revealed that GNP conjugation increased the

minimum lethal dose value of NKCT1 and partially reduced the NKCT1 induced increase of the serum biochemical tissue

injury markers. Histopathological study showed partial restoration of toxic effect in kidney tissue after GNP conjugation.

Normal lymphocyte count in culture was in the order of GNP-NKCT1>NKCT1>Indomethacine treatment. The present

study confirmed that GNP conjugation increased the antiarthritic activity and decreased toxicity profile of NKCT1.

Keywords: Gold nanoparticle, Gold nanoparticle conjugation, Naja kaouthia, NKCT1, Rheumatoid arthritis, Snake venom,

Toxicity study

Application of snake venom toxins in health and

diseases have been mentioned in folk-traditional

medicine1. Snake venom toxins showed activity against

various experimental pathology/animal studies such as

cancer2,3

, stroke4, neural trauma, alzheimer's disease,

parkinson's disease5. Anti-arthritic activity of Indian

monocellate cobra (Naja kaouthia) venom on adjuvant

induced arthritis has been reported6. Chen and

Robinson7, isolated cobrotoxin from snake Naja naja

atra, which possessed anti-nociceptive effect in mice.

Pretreatment of cobra venom factor delayed the acute

inflammation followed by induction of chronic arthritis

in rat model8. Treatment with small dose of cobra venom

factor delayed the onset of adjuvant arthritis and

inhibited the maximum inflammatory response8.

One of the major drawbacks of most of the snake

venom toxins that they failed to cross the barrier of

safety line due to their toxicity and thereby hindered

the process of drug development. In order to

overcome the toxicity of these potential toxins,

several attempts have been made which include

liposome encapsulation, silica coating and

nanoparticle tagging9,10

. Toxins have been conjugated

with nanoparticles to increase their functional

efficacy, reduction of toxicity and targeted drug

delivery11,12

. Snake venom from Walterinessia

aegyptia (WEV) was combined with silica

nanoparticles, decreased the expression of Bcl-2 and

enhanced the activation of caspase-3 in breast cancer

cell line (MDAMB-231). Nano-particle along with

WEV also showed significant reduction in actin

polymerization and cytoskeletal rearrangement13

.

Melittin (a component of bee venom) loaded

nanoparticles have the ability to inhibit infectivity by

CXCR4 and CCR5 tropic HIV-1 strains14

.

The foundation of nanotechnology research is

based on the size and shape of the nanoparticles,

where distinct optical, electronic, or magnetic

properties can be tuned during chemical synthesis.

________________

*Correspondent author

Telephone: 91-33-23508386/6387/6396/1397 (Extn: 229)

Fax: 91-33-2351-9755/2241-3288

E-mail: [email protected]

INDIAN J EXP BIOL, AUGUST 2014

764

There is an enormous interest in exploiting gold

nanoparticals (GNP) in various biomedical

applications since their size scale is similar to that of

biological molecules like protein, DNA etc. Over the

past two decades, their colours and unique

physicochemical properties have also attracted

attention due to their historical importance and current

applications in biotechnology and material science.

The use of GNP as biomarkers in biology has been

rapidly increased due to high scattering efficiencies,

immune to photo bleaching, and non-cytotoxic, which

provides advantage over semiconductor

nanoparticles15

. Functionalizing gold nanoparticles

with proteins could promote efficient binding,

clearance, and biocompatibility16

. Gold nanoparticle

conjugation is a dynamic domain of drug designing in

biomedical research and one of the most important

emerging areas in modern toxicology for effective

targeted delivery, reducing toxicity and increasing

efficacy. In the present study, a snake venom protein

toxin NKCT1, isolated from Indian monocellate

cobra, Naja kaouthia Lesson 1831, venom,

conjugated with gold nanoparticle and its anti-arthritic

property as well as toxicity profile have been

evaluated in experimental animal model.

Materials and Methods

Materials and reagents—Chemicals and reagents

used were of analytic grade. The following chemicals

were used: acetyl acetone reagent, acrylamide,

ammonium persulphate, bisacrylamide, disodium

hydrogen phosphate, cupric sulphate, ethanol, folin

ciocalteu reagent, glycine, indomethacin,

mercaptoethanol, methanol, para dimethyl amino

benzaldehyde, paraffin, P-nitrophenyl phosphate,

poly(ethylene)glycol, TRIS, sodium borohydride,

sodium lauryl sulphate were of SRL (India); BSA,

carboxymethyl cellulose, coomassie brilliant blue,

Freund’s complete adjuvant, HAuCL4 and histopaque

were from Sigma (USA); bromophenol blue, CK-MB

kit, diastix-reagent strips, LDH, methyline blue,

osteomol, potassium dihydrogen phosphate, SDS,

SGOT, SGPT kit, sodium potassium tartarate,

TEMED, urea kit and, xylene were from Merck

(India); eosin, formaldehyde and hematoxylin were

from Qualigen (India); ELISA kit - CINC1, TNF-α,

IL-1β, IL-6, CINC-1, IL-10 and VEGF kit were from

R & D (USA); Drabkin solution of Cogent (India) and

tris buffer were from Spectrochem (India).

Collection of snake venom—Lyophilized Naja

kaouthia snake venom (NKV) was purchased from

Calcutta Snake Park, Kolkata, India. Venom

concentration was expressed in terms of dry

weight/protein equivalent17

.

Purification of NKCT1—NKCT1 was purified from NKV by Ion-exchange column chromatography

and HPLC18

. The fraction was desalted and

concentrated by centricon (Millipore MWCO 3 k).

Purified NKCT1 was checked for homogeneity by

HPLC and SDS-PAGE gel electrophoresis19

.

Synthesis and characterization of GNP-NKCT1—

GNP was prepared by sodium borohydrate reduction

method20

with modification. Using poly

ethyleneglycol (PEG), NKCT1 conjugation and

characterization was done as reported earlier10

.

Experimental animals—Wistar male albino rats

(120±10 g) and Swiss albino male mice (20±2 g) were

procured from the approved animal breeders and

housed in standard polypropylene cages at controlled

temperature (25±2 °C), with light conditions (12 h light

and dark cycle) and 65±5% RH. The animals were

provided with pellet diet, green vegetables, gram and

water ad libitum. The experiments were conducted

according to the departmental animal ethics

committee for the purpose of control and supervision

of experiments on animals. All animal experiments

were approved by the Institutional Animal Ethics

Committee (AEC Ref: 820/04/ac/CPC SEA.2010).

Antiarthritic activity—Development of experimental

rheumatoid arthritis: Experimental rheumatoid arthritis

(RA) was developed by injecting (sub-plantar) 0.1 mL

emulsion of FCA in olive oil (1:1 v/v) at a

concentration of 0.25 mg heat killed Mycobacterium

tuberculosis/mL of emulsion into the right hind footpad

of male rats on day 021

.

Treatment schedule: Rats were divided into

following 6 groups of 6 each: Gr. 1: sham control;

Gr. 2: arthritis control, Gr. 3: standard (indomethacin,

0.25 mg.100g-1

x 5 days alternately, po), Gr. 4:

NKCT1treated (2µg.100g-1

x 14 days, ip), Gr. 5:

GNP-NKCT1 treated (2µg.100g-1

x 14 days, ip),

Gr. 6: GNP treated (200 µL of 48µM GNP.100g-1 x 14

days, ip). Treatment was started on day 3 after FCA

induction. The rats in the control group received the

same volume of vehicle. Urine was collected on day 14.

On day 16 blood was collected and serum was separated

for the analysis of biochemical parameters. Right hind

paw and ankle were collected for histological studies.

Physical parameters: On day 0, 2, 5, 10 and 15,

paw and ankle swellings of all animals were measured

with digital caliper (Mitutoyo, Japan). Paw and ankle

SAHA et al.: NANOGOLD CONJUGATED PROTEIN TOXIN & ANTIARTHRITIC POTENTIAL

765

diameter measured just prior to arthritis induction

were used as the control diameter (day 0).

Biochemical analysis of urine and serum: Urinary

hydroxyproline22

and glucosamine23

were measured

spectrophotometrically (Analab-180, USA). Serum

alkaline (ACP) and acid phosphatase (ALP) were

measured by biochemical methods24

. The measurements

of serum pro and anti-inflammatory interleukins

(TNF-α, IL-1β, IL-6, CINC1, IL-10, VEGF) were done

by ELISA kit, using ELISA reader (BioTek, ELx800).

Histopathological studies of joints— Joints were

fixed in 10% buffered formalin for 24 h, decalcified

in osteomol for 4-5 days, dehydrated in graded

alcohol, cleared in xylol and embedded in paraffin

wax (56–58 °C). Sections (5 µm thick) were cut with

rotary microtome (Weswox Optik, India), stained

with haematoxylin-eosin, and observed under bright

field microscope (Motic BA 450, Germany) and

photography were captured with Motic software

(Motic Images Plus 2.0 software).

Acute toxicity studies—Determination of minimum

lethal dose: NKCT1 and GNP-NKCT1 minimum

lethal dose (MLD) was determined in Swiss albino

male mice. Different dose (1, 1.5, 2, 2.5, 2.75, 3 and

3.5 mg.kg-1

) of NKCT1 and GNP-NKCT1were

administered (sc) to different animal groups and

mortality were recorded up to 24 h of observation.

The minimum dose that killed 100% animals in 24 h

was considered as the minimum lethal dose (MLD).

Acute toxicity treatment schedule: Swiss albino

male mice (20±2 g) were divided into following 3

groups of 6 each: Gr. 1: Sham control; Gr. 2: NKCT1

treated (2 µg.100g-1

, sc); Gr. 3: GNP-NKCT1 treated

(2 µg.100g-1

, sc). The toxic manifestation, mortality,

food and water intake, fecal consistency of animals

were monitored up to 24 h and qualitative urine

parameters were recorded by Diastix- Reagent Strips

(Merck, India) after 24 h.

Hematology and biochemical parameters: Blood

was collected from all the animals at 8 and 24 h by

cardiac puncture and in heparin vials. Total WBC,

RBC count and Hb% were estimated. Serum glutamate

oxaloacetate transaminase (SGOT), serum glutamate-

pyruvate transaminase (SGPT), urea, creatinine,

creatine kinase (CK) MB isoenzyme (CK-MB), lactate

dehydrogenase (LDH) were estimated after 24 h,

using biochemical kits (Ecoline, Merck, India).

Histopathology of kidney: Kidney tissue was

collected after 24 h of treatment and fixed with 10%

buffer formalin for 24 h. The organs were dehydrated

in graded alcohol, cleared in xylol and embedded in

paraffin wax (56–58 °C). Sections (5 µm thick) were

cut with rotary microtome (Weswox Optik, India),

stained with haematoxylin-eosin, and observed under

bright field microscope (Motic BA 450, Germany).

Photographs were captured with Motic software

(Motic Images Plus 2.0 software) to observe

microscopic changes in the organs.

Human lymphocytes collection and toxicity study:

Blood was collected in heparinized vial from the

healthy adults (aged 24-30 years) after informed

consent. The experiment was approved by the

Institutional human ethics committee (Ref. No. IHEC/

AG/HUM/P17/12). Lymphocytes were collected from

whole blood using histopaque after centrifugation at

1200 rpm for 5 min. They were cultured in sterile

RPMI 1640 media. Cells were grown in a CO2

incubator at 37 °C with 5% CO2 in humidified

condition. Normal lymphocytes (1x 106) were seeded

on 96 well and treated with GNP-NKCT1/NKCT1

(2 µg.mL-1

) of cell lines with respect to standard drug

Indomethacine (250 µg.mL-1

). The cell growth

inhibition studies were done using 0.2% trypan blue

with direct count under light microscope (Olympus,

Tokyo) using haemocytometer chamber.

Statistical analysis—Data were expressed as

mean±SE (n=6). The repeated measure analysis of

variance (ANOVA) was used to determine significant

differences between groups p<0.05 was considered to

be statistically significant.

Results Purification of NKCT1—Naja kaouthia venom was

applied on CM-cellulose column equilibrated with

phosphate buffer (pH 7.2). Protein fraction eluted by

0.5 M NaCl was collected and further purified by

HPLC using Protein Pak 60 column. A single sharp

peak with retention time 22 min was collected.

SDS-molecular weight of the protein was found to be

6.76 kDa and designated as NKCT1.

Synthesis and characterization of GNP-NKCT1—

Preparation of GNP-NKCT1 through sodium

borohydrate reduction method by using NKCT1,

GNP, PEG, resulted a light purple coloured colloidal

solution, stable at room temperature (25±2 °C) and

pH 7.2. Hydrodynamic size of GNP-NKCT1 was

determined by DLS was 68–122 nm with average of

92 nm10

. TEM image analysis confirmed the actual

size of GNP as 5-25 nm, where GNPs were arranged

within the protein (NKCT1) core (Fig. 1).

INDIAN J EXP BIOL, AUGUST 2014

766

Anti-arthritic activity of GNP-NKCT1

Physical parameters—Induction of RA resulted

significant increase of the paw and ankle diameter in

respect to sham control rats Gr. 1 rats (4.14±0.18 mm

and 4.91±0.13 mm). GNP-NKCT1 treatment

significantly decreased paw and ankle diameter up to

4.54±0.17 mm and 5.12±0.09 mm respectively;

NKCT1 treatment significantly decreased paw and

ankle diameter up to 5.17±0.12 mm and 5.15±0.16 mm

respectively; GNP treatment significantly decreased

paw and ankle diameter up to 5.23±0.22 mm and

5.33±0.13 mm respectively as compared to arthritic

control Gr. 2 rats (6.56±0.24 mm and 6.76±0.21 mm)

after day 15, whereas standard drug treatment

significantly decreased paw and ankle diameter up to

4.76±0.13 mm and 5.3±0.12 mm. (Fig. 2a and b).

Effect of GNP-NKCT1 on urinary parameters—

GNP-NKCT1 treatment significantly decreased

hydroxyproline (64.67%) and glucosamine (70.57%)

levels; NKCT1 treatment significantly decreased

hydroxyproline (56.18%) and glucosamine (65.87%)

levels; GNP significantly decreased hydroxyproline

(65.93%) and glucosamine (57.06%) levels as

compared with arthritic control Gr. 2 rats, whereas

standard drug treatment significantly decreased

54.32% hydroxyproline and 55.26% glucosamine

level (Fig. 2c).

Effect of GNP-NKCT1 on serum parameters—

GNP-NKCT1 treatment significantly decreased ACP

(83.28%) and ALP (54.57%) levels; NKCT1

treatment significantly decreased ACP (70.37%) and

ALP (53.14%) levels; GNP treatment significantly

decreased ACP (67.6%) and ALP (50.49%) levels as

compared to arthritic control Gr. 2 rats, whereas

standard drug treatment significantly decreased

59.35% ACP and 60.01% ALP level (Table 1).

Effect of GNP-NKCT1 on serum cytokines—GNP-

NKCT1 treatment significantly decreased TNF-α

(33.12%), IL-1β (34.06%), IL-6 (71.79%), CINC1

(40.76%) and VEGF (48.66%) level; NKCT1

treatment significantly decreased TNF-α (28.23%),

IL-1β (21.97%), IL-6 (47.39%), CINC1 (27.29%) and

VEGF (29.69%) level; GNP treatment significantly

decreased TNF-α (21.54%), IL-1β (19.18%), IL-6

(51.32%), CINC1 (34.13%) and VEGF (49.35%)

level as compared to arthritic control Gr. 2 rats,

whereas standard drug treatment significantly

decreased 37.17% TNF-α, 32.47% IL-1β, 75.23%

IL-6, 35.18% CINC1 and 27.53% VEGF level

(Fig. 2d, Table 1).

GNP-NKCT1, NKCT1 and GNP treatment

significantly increased serum anti-inflammatory

cytokine IL-10 level (30.54, 20 and 20.46%) as

compared with arthritis control Gr. 2 rats, whereas

Fig. 1—Transmission electron microscopy and DLS study of

(a) only GNP, size 2–8 nm with average size of 4 nm (b) GNP-

NKCT1 where GNP were arranged within protein core, size was

of 68-122 nm with an average of 92 nm

SAHA et al.: NANOGOLD CONJUGATED PROTEIN TOXIN & ANTIARTHRITIC POTENTIAL

767

standard drug treatment showed 15.5% increase in

IL-10 level (Table 1).

Effect of GNP-NKCT1 on joint histopathology—

Arthritic control Gr. 2 rats showed cellular

infiltration, increased destruction of synovial

membrane, decreased synovial space of joint

histology, whereas GNP-NKCT1, NKCT1 and GNP

treated groups showed partial restoration of normal

architecture of joint histology as compared with

arthritis control Gr. 2 rats.

Table 1—Effect of GNP-NKCT1 and NKCT1 serum parameters of FCA induced arthritis in male albino rat

[Values are mean±SE from 6 observations each]

Animal group ACP

(µmol of PNP. min-1)

ALP (µmol of

PNP. min-1)

IL-10

(pg. mL-1)

VEGF

(pg. mL-1)

Gr. 1 16.21±2.50 60.67±4.04 212.18±4.80 298.67±15.21

Gr. 2 97.45±4.83** 159.67±6.12** 156.58±8.60** 686.24±15.96**

Gr. 3 39.61±2.90* 63.38±4.41* 180.85±6.77* 497.32±13.84*

Gr. 4 28.87±2.78* 74.82±9.47* 187.90±14.04* 428.62±11.41*

Gr. 5 16.29±3.90* # 72.53±10.49* 204.40±13.83*# 352.28±11.16*#

Gr. 6 31.57±1.67* 79.04±5.21* 188.63±11.48* 347.53±11.76*

P values: **< 0.05 compared with sham control group. *< 0.05 compared with RA control group. #< 0.05 compared with NKCT1 treated

group. Gr. 1= Sham control, Gr. 2= arthritis control, Gr. 3= standard drug (indomethacin), Gr. 4= NKCT1 treated (2µg.100g-1 × 14 days,

ip), Gr. 5= GNP-NKCT1 treated (2µg.100g-1 × 14 days, ip), Gr. 6= GNP treated (200 µL of 48µM GNP.

100g-1 × 14 days, ip)

Fig. 2—Effect of NKCT1, GNP-NKCT1 and standard drug on (a) paw diameter (b) ankle diameter (c) urinary

hydroxyproline/glucosamine and (d) serum interleukins levels of FCA induced arthritis in male albino rats. Values are mean±SE from

6 observations each. Analysis were done using one way ANOVA, P values: *< 0.05 compared with arthritic control group.**< 0.05)

compared with sham control group. ***< 0.05 compared with NKCT1 treated group

INDIAN J EXP BIOL, AUGUST 2014

768

Acute toxicity of GNP-NKCT1 Determination of minimum lethal dose (MLD)—

The minimum lethal dose (MLD) of GNP-NKCT1

and NKCT1 was found to be 3 and 1.5 mg.k

-1 (sc)

respectively in Swiss albino male mice.

No animal mortality was recorded after 24 h of

treatment. Fecal consistency, food and water intake

were normal in all three groups. After 24 h of

treatment, urine strip test revealed no change in

urinary glucose, bilirubin, ketone, specific gravity,

blood, pH, urobilinogen in GNP-NKCT1 and NKCT1

treated mice. However, NKCT1 treated Gr. 2 mice

urine showed presence of protein (+++) as compared

with GNP-NKCT1 treated Gr. 3 mice urine protein

(+) (Fig. 3a).

Effect on hematological parameters—A significant

increase in WBC count was observed in NKCT1

(80.21%) and GNP-NKCT1 (40.6%) treated mice,

when compared with sham control Gr. 1 mice. No

significant change in total RBC count and Hb% was

observed in NKCT1 and GNP-NKCT1 treated group

of mice as compared to sham control Gr. 1 mice.

Serum biochemical parameters—There was a

significant increase in serum creatinine, urea, CK-MB

and LDH (136.1, 154.9, 75.14 and 51.2%) due to

NKCT1 treated Gr. 2 mice as compared with sham

control Gr. 1 mice. GNP-NKCT1 treatment also

significantly increased serum urea, CK-MB and LDH

levels (46.2, 34.28 and 16.6%) as compared with sham

control Gr. 1 mice. No significant change in serum

creatinine level was observed after GNP-NKCT1

treatment. (Table 2).

Histopathological changes—NKCT1 treated

kidney histology showed partial tubular and

glomerular necrosis and increased capsular space.

Whereas GNP-NKC1 treatment showed less tubular

and glomerular necrotic effect as compared with

NKCT1 treated kidney tissue (Fig. 3b).

Toxicity studies on human lymphocytes—NKCT1

(2 µg.mL-1

), GNP-NKCT1 (2 µg.mL-1

) treatment

decreased the lymphocyte count (28-30 and 16-18%)

as compared with the standard drug Indomethacine

(44-46%).

Table 2—Effect of GNP-NKCT1 and NKCT1 on serum biochemical

parameters of male albino mice.

[Values are mean±SE from 6 observations each]

Animal

group

Creatinine

( mg.dL-1

)

Urea

( mg.dL-1

)

LDH

(U.L-1

)

CK-MB

(IU.L-1

)

Gr. 1 0.83±0.06 28.04±2.01 149.37±7.56 10.67±1.14

Gr. 2 1.96±0.08* 71.48±5.02* 224.14±10.24* 18.71±6.54*

Gr. 3 0.98±0.03*# 40.76±1.48*# 173.83±7.67*# 14.21±2.78*#

P values: *< 0.05 when compared with sham control group. #< 0.05

when compared with NKCT1 treated group. Gr. 1= sham control,

Gr. 2= NKCT1 treated mice, Gr. 3= GNP-NKCT1 treated mice.

Fig. 3— (a): Effect of GNP-NKCT1 and NKCT1 on male albino

mice urinary protein, analyzed by Multistix urine strip b(i), Blank

strip; b(ii), absence of protein in control mice urine;

b(iii), presence of protein (+) in GNP-NKCT1 treated mice urine;

b(iv), presence of protein (+++) in NKCT1 treated mice urine.

(b): Histopathological changes induced by of GNP-NKCT1 and

NKCT1 on male albino mice kidney. b(i), control mice kidney;

b(ii), GNP-NKCT1 treated mice kidney; b(iii and iv), NKCT1

treated mice kidney. Arrows show the glomerular necrosis and

increased capsular space, indicates the nephro-toxicity of NKCT1.

SAHA et al.: NANOGOLD CONJUGATED PROTEIN TOXIN & ANTIARTHRITIC POTENTIAL

769

Discussion In this study, a cytotoxic snake venom protein

toxin NKCT1 was tagged with gold nanoparticles

with a poly ethylene glycol (PEG) ligand protecting

layer. It had been demonstrated that PEG ligands were

very much effective in preventing nonspecific binding

of peptides, proteins and enzymes to the gold

nanoparticle surfaces25

. PEG had been extensively

used as a biomaterial because of its low toxicity, low

immunogenicity26

. PEG coated GNP with functional

carboxyl groups on their surface was formed and

allowed for further controlled conjugation with

NKCT1 that avoided peptide-peptide interaction and

aggregation within the nanoparticles. It was

previously reported that, GNP up to 100 nm size was

easily taken inside the cells and trapped within the

vesicles in cytoplasm27

. The hydrodynamic size of

GNP-NKCT1 obtained by DLS was 68-122 nm with

average of 92 nm, confirmed the effective size of

GNP-NKCT1 for cellular uptake. During the

assessment of anti-rheumatic activity, GNP-NKCT1

(total 28 µg of NKCT1 and 26.5 µg of GNP) was

administrated per rat during the treatment period. Paw

and ankle swelling of FCA induced rats were

significantly reduced after GNP-NKCT1 treatment as

compared with the arthritic control Gr. 2 rats. It was

reported that infiltrated pro-inflammatory cells like

neutrophils attach to the cartilage and invade the

cartilage matrix by its lysosomal enzymes and

oxygen-derived reactive species28

. It was very likely

that the inhibition of hind paw and ankle swellings by

GNP-NKCT1 treatment was due to inhibition of pro-

inflammatory cells infiltration and reducing tissue

destruction. It was further supported by the reduction

of chemo attractant CINC-1 level by GNP-NKCT1

treatment in arthritic rats.

Collagen and cartilage matrix glycoproteins having

high content of hydroxyproline and glucosamine.

Catabolism of insoluble collagen and cartilage matrix

glycoproteins is the major source of urinary

hydroxyproline and glucosamine29

that increases or

decreases in a number of clinical conditions30

including Paget's disease of bone, carcinoma with

metastasis in bone, osteoporosis, arthritis etc31,32,3

.

Increased activities of matrix metalloproteinease

enzymes and collagen degradation were responsible

for the elevated levels of hydroxyproline in arthritic

rat urine33

. Significant restoration of urinary

hydroxyproline and glucosamine by GNP-NKCT1

treatment indicated that GNP-NKCT1 provided

condroprotection by inhibiting the degradation of

collagen and cartilage matrix. Acid and alkaline

phosphatases (ACP and ALP) activity is a measure of

lysosomal integrity. In adjuvant-induced arthritis and

inflammatiory disorders, an increase of ACP and ALP

level was observed due to their release from the

degraded cell lysosomes in the area of inflammation,

which facilitated the degradation of extra cellular

matrix of synovium3. GNP-NKCT1 treatment

significantly decreased the ACP and ALP levels in

FCA induced rats, which indicated that GNP-NKCT1

possess the potential to restore the damages of

lysosomal membrane integrity.

In animal models, inhibition of TNF-α result in

decreased inflammation, while inhibition of IL-1β

effectively prevented cartilage destruction34

.

Similarly, IL-6-deficient mice did not develop bone

erosion35

. Therapeutic inhibition of IL-6 may reduce

generalized osteoporosis, joint inflammation and local

joint destruction in RA36

. The direct involvement of

TNF-α and other pro-inflammatory cytokines in the

development and maintenance of RA suggested that

blocking of their expression in the synovial tissue

could be beneficial for therapy. TNF-α and other

cytokine synthesis is known to be regulated at both

the transcriptional and post transcriptional level. It

was observed that GNP-NKCT1 treatment

significantly reduced the pro-inflammatory cytokines

like TNF-α, IL-1β, IL-6 in respect to arthritic control

rats. Synthesis of those cytokines were regulated in

the transcription level by transcription factor NF-kβ in

pathogenesis of cancer and inflammatory disorders37

.

It was observed that GNP-NKCT1 inhibited the NF-

kβ activation in cancer cells (Gomes et al,

unpublished data). Therefore, antiarthritic property of

the GNP-NKCT1 may attributed to its’ indirect mode

of action, mainly by blocking the upstream regulation

of NF-kβ signaling pathway and synthesis of pro-

inflammatory cytokines. CINC-1, a pro-inflammatory

cytokine promotes recruitment of neutrophil in the

affected joints and helps to progress inflammation38

. It

was also observed that GNP-NKCT1 treatment

antagonized the action of CINC-1. An increased

production of IL-10 by non-T cells was reported in

patients with RA39,40

. There was an initial burst of

pro-inflammatory cytokines (TNF-α, IL1, IL6)

followed by a rise in IL10 synthesis since IL10

production requires both the synthesis of TNF-α and

IL141

. It was found that GNP-NKCT1 treatment

significantly increased the anti-inflammatory cytokine

INDIAN J EXP BIOL, AUGUST 2014

770

IL-10 level in arthritic rats. It may contribute to

diminish T cell mediate inflammation and increased

antibody production in RA.

Beside the onset of inflammation, angiogenesis is a

very important event that provides the vascular

network, necessary to sustain synovial and immune

cell proliferation in RA. VEGF, the key regulatory

molecule of angiogenesis, induced the proliferation

and migration of vascular endothelial cells during the

progression of new blood vessels formation in RA42

.

It was observed that GNP-NKCT1 restored the serum

VEGF level, which was significantly increased in

arthritic rat. Significant reduction of serum VEGF

level by GNP-NKCT1 treatment in arthritic rats

indicate it’s antiangiogenic property, that may

attributed due to the presence of GNP, because GNP

treatment itself significantly reduced the VEGF level

in arthritic rats. Joint histology showed efficacy of

GNP-NKCT1 for partial restoration of synovial space

and the normal joint architecture.

Debnath et al.18

showed that NKCT1 shared

toxicity along with the anticancer activity and the

toxicity being one of the major drawback of this

molecule. One of the major target of the present

study, how to minimize the toxicity of NKCT1?

Whether nanogold conjugation would be able to alter

the toxicity of NKCT1? The toxicity data on GNP-

NKCT1 clearly showed that the toxicity of NKCT1

was reduced due to nanogold conjugation as observed

by the reduced lethal action in mice. In the acute

toxicity studies, several biochemical markers of major

organs (heart, kidney) were increased due to NKCT1

treatment, which were significantly low in GNP-

NKCT1 treated animals. NKCT1 was nephrotoxic in

nature as showed by the presence of protein (+++) in

the urine of NKCT1 treated animals. Low amount of

protein (+) in the urine of GNP-NKCT1 treated

animals confirmed that the nephrotoxic activity of

NKCT1 was minimized by nanogold conjugation thus

making the molecule less toxic. Histopathological

observation of the kidney also confirmed the above

findings.

NKCT1 induced myotoxicity markers (CK-MB,

LDH) were significantly low in GNP-NKCT1 treated

mice, thus the myotoxic action of NKCT1 was also

reduced by nanogold conjugation. In vitro culture of

lymphocyte showed that nanogold conjugated

NKCT1 was less toxic than the NKCT1 and

indomethacine. It is likely that gold conjugation

masked the protein part of NKCT1 responsible for the

toxicity manifestation or inactivated the toxic moiety

of NKCT1 by certain chemical bonding. Tagging of

GNPs with protein/peptide prolongs the circulation

time, thus offering an increased probability of

accumulation in the abnormal cells and targeted

binding to sites of angiogenesis or infection12

. The

delivery of conjugated protein/peptide is small and

decreased in nonspecific targeting attributable to the

use of GNPs. Moreover, pegylation of GNPs

prevented their interaction and uptake by normal

lymphocytic cell or endothelial cell14,10

. Fortuitously

extra loaded GNPs that do not reach their targets are

accumulated and deactivated in the liver12

, while

thermodynamically favorable GNP-NKCT1 (neutral

GNP charged with cationic protein) reach their targets

and engulfed by enhanced phagocytotic abnormal

cells that leads to cell death.

In the present study it was observed that, urinary

serum biochemical markers, pro-inflammatory

cytokines levels were significantly reduced after

GNP-NKCT1 and NKCT1 treatment. However the

levels of those arthritic markers (hydroxiproline,

glucosamine, ACP, IL-1B, IL-6, CINC1, VEGF) were

significantly low in GNP conjugated NKCT1 treated

rats than that of unconjugated NKCT1 treated rats.

GNP-NKCT1 significantly increased in anti-

inflammatory cytokines IL-10 levels than that of

unconjugated NKCT1 treatment. Furthermore present

study also showed that GNP conjugation reduced the

toxicity profile of NKCT1 along with increasing it’s

antiarthritic efficiency. Serum level of tissue injury

markers (creatinine, urea, LDH, CK-MB) were found

to be significantly low in GNP-NKCT1 treated mice

than that of NKCT1 treated mice group. Recently it

had been reported that GNP conjugation reduces the

cardio, neuro and nephrotoxic limitations of the

NKCT143

. Therefore GNP conjugation not only

increased the antiarthritic property of NKCT1 but also

beneficial for the reduction of the toxicity. Further

detail studies on this area are warranted for

understanding the mechanism of reduction of toxicity

and antiarthritic potential of GNP-NKCT1 for

biomedical application in the near future.

Conclusion This study confirmed a successful conjugation of

gold nanoparticle with NKCT1, a cytotoxin from

Naja kaouthia snake venom which showed increased

antiarthritic potential and lower toxicity profile

through in vivo and in vitro experiments. Further

SAHA et al.: NANOGOLD CONJUGATED PROTEIN TOXIN & ANTIARTHRITIC POTENTIAL

771

studies on the molecular mechanism of anti-arthritic

activities of GNP-NKCT1 and its toxicity are in

progress.

Conflict of interest

No conflict of interest exist among the authors.

Acknowledgement This research work was sponsored by Department

of Biotechnology, Govt of India, New Delhi

(Ref. no. BT/PR14811/NNT/28/500/2010).

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