Introduction: the pain and swelling of rheumatoid arthritis, and

Introduction:

Gold is a chemical element with symbol Au (from Latin: aurum) and atomic number 79. In its purest form, it is a bright,
slightly reddish yellow, dense, soft, malleable and ductile metal.
The pure metal melts at 1063°C and boils at 2966°C.It has an atomic weight of
196.967 with a density of 19.32 g cm?3
at 20°C. Its beauty and rarity has led to the use in jewelry and in coinage. Chemically, gold is a transition metal and a group 11 element. Common oxidation state of
gold is +III and +I, although it can show oxidation states from –I to + V. It
is one of the least reactive chemical elements, and is solid under standard conditions. The metal therefore occurs
often in free elemental (native) form. Gold resists attacks by individual acids, but it can be dissolved by aqua regia (1:3 mixture of nitric acid and
hydrochloric acid). The acid mixture causes the formation of a soluble gold tetrachloride anion. Gold metal also dissolves
in alkaline solutions of cyanide, which are used in mining and electroplating. It is insoluble in nitric acid, which dissolves silver and base metals.
Its insolubility in nitric acid is used to refine it from silver or other base
metals.

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Gold because of its malleability and
ductility is used in various purposes such as infra-red shields, heat resistant
suits.

Gold as we know is probably the most ancient
administered medicine.  In the 19th century gold was used as a “nervine,” therapy for
nervous disorders. Depression, epilepsy, migraine, and glandular problems such as amenorrhea and impotence were treated with the help of gold. 1

Some gold salts do have anti-inflammatory properties and at present two are still
used as pharmaceuticals in the treatment of arthritis and other similar
conditions in the US (sodium aurothiomalate and auranofin). These drugs have been explored as a
means to help to reduce the pain and swelling of rheumatoid arthritis, and also (historically) against tuberculosis and some parasites 2

 Gold Nanoparticles because of their unique
optical, electronical and molecular recognition properties are used widely in
biological fields for various purposes.  Nanoparticles can be produced through various methods. Chemical process
are the most popular methods for nanoparticle production but some of the
chemical processes use toxic chemicals and hence can not be used for biological
purposes. Synthesis of Au nanoparticles using microbial, plant, plant extracts
and enzymes are the suggested eco-friendly ways. There are many microbes which
are known to produce nanostructured mineral crystals and metallic nanoparticles
with properties similar to chemically synthesised materials, while exercising
strict control over size, shape and composition of the particles.

 A fungus
Verticillium sp. when exposed to aqueous AuCl4? ions results in reduction of the metal
ions and formation of gold nanoparticles of around 20 nm diameter.3 The gold nanoparticles formed are reported to be on both the surface and
within the fungal cells (on the cytoplasmic membrane) with negligible reduction
of the metal ions in solution.4

Many
bacteria such as Thermomonospora sp, 5  Rhodococcus sp. , Rhodopseudomonas
capsulate ,6
Pseudomonas aeruginosa, 7Delftia acidovorans 8 produce gold nanoparticles by the process of
reduction of gold salt. Rhodococcus sp. is reported to produce gold
nanoparticle intracellularly 9 while others produce extracellularly.

Plant
extract have also been used for the preparation of gold nanoparticle. For
example, extract of Cymbopogon flexuosus form
nanoparticles extracellularly 10 while live alfalfa plant produce intracellularly 11.  One of the advantage of using plant for
nanoparticle production instead of using bacteria and fungi is the lack of
pathogenicity of plant nanoparticles. These organisms probably form
nanoparticles as a surviving mechanism adapted by the organism to cope with the
high levels of metal in the environment. The mechanisms may involve alteration
of the chemical nature of the organisms so that the metal no longer causes
toxicity. In this process the metal can be reduced and nanoparticle can be
produced. Thus, it can be said that the production of nanoparticle is
by-product of resistance mechanism evolved by the organism against the specific
metal, and therefore this process is exploited for the green synthesis of
nanoparticle.

To
date, there are numerous techniques for synthesizing nanoparticles. However,
these techniques fall into two broad approaches and can be defined as either a
top down approach or a bottom up approach 12. The top down approach
starts with a material of interest, which then undergoes size reduction via
physical and chemical processes to produce nanoparticles. Importantly,
nanoparticles are highly dependent on their size, shape, and surface structure
and processing tends to introduce surface imperfections. These surface
imperfections can significantly impact on the overall nanoparticle surface
physicochemical properties 13. In the bottom up approach, nanoparticles
are built from atoms, molecules and smaller
particles/monomers 1415 16. In either approach, the
resulting nanoparticles are characterized using various
techniques to determine properties such as
particle size, size distribution, shape, and surface area. This is of
particular importance if the properties of nanoparticles
need to be homogeneous for a particular application. 17

During
nanoparticle synthesis the gold salt is reduced by any reducing agent whether
chemical or biological which results into change in colour. This is the first
qualitative indication that the nanoparticle is formed. For further study of
nanoparticles various spectroscopy and microscopy techniques are being used
such as UV-Visible spectroscopy, dynamic light scattering (DLS), atomic force
microscopy (AFM), transmission electron microscopy (TEM),
scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS),
powder X-ray diffraction (XRD), Fourier transform infrared spectroscopy
(FT-IR), and Raman spectroscopy. Microscopy based techniques
such as AFM, SEM and TEM obtain data from images taken of the nanoparticles. In
particular, both SEM and TEM have been extensively used to determine size and
morphological features of nanoparticles. While spectroscopy based techniques
such as UV-vis, DLS, XRD, EDS, FT-IR, determine data related to composition,
structure, crystal phase and properties of nanoparticles. In case UV-Visible spectroscopy
wavelengths between 300 and 800 nm are generally used for characterizing
metallic nanoparticles ranging in size from 2 nm up to around 100 nm 18. For
example, gold (Au) nanoparticles are generally detected by the presence of
peaks between 500 and 550 nm.19

 DLS spectroscopy is used to determine size
distribution and quantify the surface charge of nanoparticles suspended in a
liquid.
19 20

Researchers
have shown that different pH can also affect the formation of nanoparticles.
Importance of pH in the biosynthesis of colloidal gold using alfalfa biomass
have been shown and it is being concluded that the size of nanoparticles
changes with the change in pH. 21 Large nanoparticles are produced at lower
pH(2-4) 22.

 In case of Avena
sativa, it has been shown that formation of gold nanoparticle is highly
dependent on the pH value. At pH 2, large-sized nanoparticles (25?85 nm) are
reported, although in low quantity. At pH 3 and 4, smaller-sized nanoparticles
in a large quantity are reported. They speculated that at low pH (pH 2), the
gold nanoparticles prefer to aggregate to form larger nanoparticles rather than
to nucleate and form new nanoparticles. In contrast, at pH 3 and 4, more
functional groups (carbonyl and hydroxyl) are available for gold binding; thus,
a higher number of new Au (III) complexes would bind to the biomass at the same
time that will nucleate separately and form nanoparticles of relatively small
size. 23

Temperature
can also affect the synthesis of nanoparticle. It is reported that at higher
temperatures rate of formation of nanoparticle increases. 24 They
have also reported synthesis of nanorod and platelet-shaped gold nanoparticles
at higher temperatures and formation of spherical-shaped nanoparticles at lower
temperatures. Therefore, it can be concluded that temperature is also one of
the crucial factors which determine the size and shape of nanoparticles.

The
exact mechanism how the nanoparticles are formed is not understood still. It is
required to understand so that controlled and definite size and shaped
nanoparticle can be synthesized. 

 

 

Objectives :

1.      
To screen small biomolecules which can form AuNp.

2.      
To understand how biomolecules are effecting the formation of AuNp.

3.      
To understand how the AuNp formed are degraded when given to
biological system.

Material and Method:

1.      
HAuCl4

HAuCl4 and all the nucleotides and nucleosides – Adenosine,
AMP, ADP,ATP, Cytosine, CMP, CDP, CTP,Guanosine, GMP, GDP , GTP, Uridine,
UMP,UDP and UTP were bought from Sigma Aldrich.

Synthesis of AuNP:

AuNp was synthesized by mixing HAuCl4 with the respective nucleotide/
nucleoside and then mix it for 5 mins.

Characterization of Gold Nanoparticle

UV-vis
spectrophotometer

The reduction of pure HAuCl4 was
monitored using UV-vis spectroscopy. The colloid Au solution was scanned
using a UV spectrophotometer (Spectrum
X-5).

Transmission
electron microscopy

Microscopic image
of Au-NPs was studied using Transmission
electron microscopy (TEM). Analysis of the sample was done using a Technai TM G2 spirit (FEI, The Netherlands) with Gatan CCD
digital camera operated at an accelerating voltage of 80 kV. A drop of the solution was placed on carbon-coated
copper grid. The grid was then dried in desiccators by keeping
overnight at room temperature.

Scanning electron microscopy

SEM analysis of the sample was done using Quanta
450 (FEG, FEI, The Neetherlands).

Zeta
potential and Size measurement

Zeta potential (?)
and surface conductivity (?sc) were measured using a Malvern Nano ZS
instrument using phase analysis light scattering technique. Diluted samples
were sonicated for 5 min prior to measurements.

Results:

      1. AuNp prepared with
Nucleotides and nucleosides

HAuCL4 with Adenosine, AMP, ADP,ATP, Guanosine, GMP, GDP and GTP
changes colour within 5 mins indicating the formation of AuNp. (Fig. 1 a)
Au-GDP and GTP shows peak at 460 nm (Fig. 1 b)

Fig.1 (a) Au Nanoparticle prepared with different nucleotides and
nucleosides( Adenosine, Cytidine, Guanosine, Uridine, AMP, CMP, GMP,
UMP,ADP,CDP,GDP,UDP,ATP,CTP,GTP,UTP).

(b) UV spectra of Au nanoparticles produced with different nucleotides
and nucleosides

DLS measurement:

Sample
name

Z-Average
d.nm.

Pdi

A

872.8

0.568

C

3641

0.973

G

2065

0.744

U

1.31E+04

0.709

AMP

5401

0.883

CMP

6005

0.968

GMP

1898

0.985

UMP

6389

1

ADP

1863

0.783

CDP

7810

0.947

GDP

1699

0.869

UDP

1906

1

ATP

649

0.532

CTP

1721

0.858

GTP

908.7

0.59

UTP

2748

0.835

 

 

 

 

 

GDP-AuNP: functioning
as a UV- Sensor

GDP-AuNP
 

Au
(C)

Au
(C)

Au
(C)

GDP-AuNP
 

GDP-AuNP
 

UV

                            

                             

Fig-2.
a

GDP-AuNp
decolourises when subjected to heat ( Table 2). The decolourised solution is
stable at 4? but it changes to pink colour when exposed to UV light.
(Fig-2.a)

UV
Spectra  shows that the GDP-AuNp prepared(immediately)
gives peak at 490 nm showing the presence of nanocluster. but after treatment
of UV, a distinct peak at 550 is observed which indicates the presence of AuNp.
Fig.-2.b.

Zeta
potential of GDP_AuNp (Table-1) shows that the nanoparticle after UV exposure
are stable. Their average size ranges from 185nm to 73 nm to 59 nm
respectively. Their polydispersity index is 0.2 to 0.4 and 0.4 respectively.

                                                             

Fig. 2.b

Estimation of hydrodynamic radius and charge by DLS.

SAMPLE

GDP-AuNp (freshly prepared)

GDP-AuNP (decolourised)

GDP-AuNp 
(UV treated)

DLS

Z-Average (d.nm)

185.1

73.51

59.64

PDI

0.230

0.404

0.44

ZETA POTENTIAL (mV)

-16.8

-29.1

-37.5

Table-1

Time required by GDP-AuNp to decolourise after their treatment with
temperature.

TEMPERATURE ?C

TIME
REQUIRED FOR DECOLOURISATION

30

33 hours

34.8

15 hours

39.3

10 hours

45.3

4 hours

49.9

3 hours

53

2 hours 30
mins

55

1 hour 50
mins

59.9

50 mins

64.3

45 mins

70.3

28 mins

75

20 mins

78.1

14 mins

80

11 mins

Table-2

TEM, SEM of GDP-AuNP

TEM shows that the size of GDP-AuNP (prepared
immediately) ranges from 1.52 nm to 4.0 nm while that of GDP-AuNp after
decolourisation is 3-4nm and after UV exposure it is                   Fig.-3

1-Initial

     

      

 

                  

1.      
After decolourisation

           

3-
After treating with UV

       

   

 
Fig.-3    
    

Effect of different pH of HAuCl4 on GDP-AuNp
formation:

With increase of pH colour intensifies till ph 5 and
then starts declining. Fig.4.a. and UV spectra shows increasing curve till ph 7
and then it also starts declining.

Zeta potential shows that except for GDP-AuNp at pH 1
all are stable. Table -3.

 

Control pH 1   
2       3         4        5         6       7             8        9     10

 

Fig.4 a

Fig.4.b.

Estimation of hydrodynamic radius and surface charge with the help of
DLS

   Sample Name

    Z-Average (d.nm)

     Pdi

    Zeta potential

HAuCL4  pH1

315.4

0.445

-20.3

HAuCL4  pH2

401.3

0.385

-37.9

HAuCL4  pH3

157

0.219

-44.9

HAuCL4  pH4

88

0.243

-36.1

HAuCL4  pH5

76.73

0.24

-46.6

HAuCL4  pH6

251.5

0.329

-52.3

HAuCL4  pH7

161.6

0.227

-48

HAuCL4  pH8

128.5

0.207

-49.5

HAuCL4  pH9

222.4

0.287

-20.6

HAuCL4  pH10

238.9

0.381

-53.9

Table-3

 

Effect of different conc. of HAuCL4 on GDP- AuNp
formation:

By increasing and
decreasing the concentration of HAuCl4, there is no effect on colour but UV
Spectra shows distinct peak at 440 nm (10 mM conc.).Fig. 5.a,b.  Zeta potential shows that GDP-AuNp prepared
with effective conc. of HAuCl4 -10mM is the most stable. Table -4

 

GDP
(C)

HAuCl4 (mM)                        25          10           5

Fig.5.a.

Fig.5.b.

Estimation of hydrodynamic radius and surface charge with the help of
DLS

  Sample Name

     Z-Average         (d.nm) 

Pdi

         Zeta potential

HAuCL4 25 mM

115.7

0.076

-38.8

HAuCl4 10 mM

856.6

0.77

-51

HAuCl4 5 mM

63.37

0.144

-23.7

Table-4.

DISCUSSION:

HAuCL4 when treated with Adenosine, AMP, ADP, ATP,
Guanosine, GMP, GDP and GTP changes colour within 3-5 mins. indicating
formation of gold nanoparticle. But GDP nanoparticle when subjected to heat for
a specific period of time becomes colourless and when this colourless solution
is exposed to UV, it changes to pink solution. Zeta potential shows that the
nanoparticle after UV exposure becomes stable.

TEM shows that the size of nanoparticle in all the three
cases is less than the 10 nm. Increase in pH of HAuCl4 till pH 5 stabilizes the
the nanoparticle but above that stability starts declining.

Nanoparticle prepared by HAuCl4 with effective concentration
of 10 mM was most stable, increasing or decreasing the concentration led to
instability.