2328 Int J Pharm Sci Nanotech Vol 7; Issue 1  JanuaryMarch 2014

International Journal of Pharmaceutical Sciences and Nanotechnology

Volume 7 Issue 1 January – March 2014
Review Article
IJPSN-4-25-13-BHOSALE

Innovative Eco-friendly Approaches for Green Synthesis of

Silver Nanoparticles
R.R. Bhosale*, A.S. Kulkarni, S.S. Gilda, N.H. Aloorkar, R.A. Osmani and B.R. Harkare
Department of Pharmaceutics, Satara College of Pharmacy, Satara, Maharashtra, India.
Received April 25, 2013; accepted August 13, 2013

ABSTRACT
Nanotechnology is an escalating field that has made its
contribution to all spheres of human life. The green
synthesis of nanoparticles has paved for better
methodologies and approaches in the medicinal field.
Nowadays silver, gold and other metallic nanoparticles are
used as an efficient carrier for drug molecules for
developing novel drug delivery systems. In course of
synthesizing these nanoparticles various chemicals,
solvents and reagents are used which harms our eco
system directly or indirectly. Silver nanoparticles (Ag NPs)
have been widely used as a novel therapeutic agent
extending its use as antibacterial, antifungal, anti-viral and
anti-inflammatory agent. Silver nanoparticles (Ag NPs)
prepared by green synthesis have many advantages over
conventional methods involving chemical agents associated
with environmental toxicity. Green synthetic methods
include polysaccharide method, irradiation method,
biological method, polyoxometallates method and tollens
method. Green synthesis of nanoparticles is found to be an
emerging branch of nanotechnology. The use of
environmentally benign materials like plant leaf extract for
the synthesis of nanoparticles offers numerous benefits of
eco-friendliness and compatibility for pharmaceutical and
biomedical applications as they do not use toxic chemicals
in the synthesis protocols. Rapid and green synthetic
methods using various plant extracts have shown a great
potential in silver nanoparticles (Ag NPs) synthesis. This
review article describes the bio-inspired synthesis of
nanoparticles that provides advancement over chemical
and physical methods as it is cost effective, eco-friendly and
more effective in a variety of applications.

KEYWORDS: Nanotechnology; nanoparticles; silver nanoparticles; green synthesis.

Introduction Nanotechnology is mainly concerned with synthesis of

nanoparticles of variable sizes, shapes, chemical
The term “nanotechnology” has been coined by Norino
compositions and controlled dispersity and with their
Taniguchi, a researcher at university of Tokyo, Japan in
potential use for human benefits (Elumalai et al., 2010).
1974. Nanotechnology is defined as a research for the
The field of nanotechnology is one of the most active
design, synthesis and manipulation of structure of
areas of research in modern material sciences.
particles with dimension smaller than 100 nm
Nanoparticles exhibit completely new or improved
(Sadowski, 2010). Nanoparticles are sub-nanosized
properties based on specific characteristics such as size,
colloidal structures composed of synthetic or semi
distribution and morphology. Nanotechnology is a field
synthetic polymers (Vyas and Khar, 2004). Over the past
that is burgeoning day by day, making an impact in all
few years, the synthesis of nanoparticles is an important
spheres of human life. New applications of nanoparticles
topic of research in modern material science due to their
and nanomaterials are emerging rapidly. To date,
distinctive potential applications in the field of electronic,
metallic nanoparticles are mostly prepared from noble
magnetic, optoelectronic, information storage, recording
metals i.e., Silver, Platinum, Gold and Paledium. Among
media, sensing devices, catalysis, medicine and drug
these, the noble metal silver (Ag) is the metal of choice in
delivery (Mallikarjuna et al., 2011). Nanotechnology
the field of biological system, living organisms and
provides the ability to engineer the properties of
medicine (Sudhalakshmi et al., 2011).
materials by controlling their size, and this has driven
research towards a multitude of potential uses for
Significance of Nanoparticles
nanomaterials. The reduction of materials dimension has
pronounced effects on the physical properties that may One of the first and most natural questions to ask
be significantly different from the corresponding bulk when starting to deal with nanoparticles is: “Why are
material (Benjamin and Bharathwaj, 2011). nanoparticles so interesting?” and why even bother to

ABBREVIATIONS: MMC: Ag NPs: Silver Nanoparticles; GN: Green Nanotechnology; NS: Nanosilver; POMs: Polyoxometalates;
HA: Hydroxyapatite.

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Bhosale et al: Innovative Eco-friendly Approaches for Green Synthesis of Silver Nanoparticles
work with these extremely small structures when
handling and synthesis is much more complicated than
that of their macroscopic counterparts. The answer lies
in the nature of and unique properties possessed by
nanostructures. In our macroscopic everyday experience
such phenomena as light act in an easily predictive way.
Light directed at a surface is reflected just at the angle
and with the color that would be expected. This easily
predictive behavior changes dramatically when the
reflecting particles become much smaller than the
wavelength of the incident light. Nanoparticles possess a
very high surface to volume ratio. This can be utilized in
areas where high surface areas are critical for success.
This could for example be in the catalytic industry; some
nanoparticles actually have proven to be good catalysts.
Some nanoparticles also show bactericidal effects and
here a high surface to volume ratio is also important.
Nanoparticles are often in the range 10-100 nm and this
is the size as that of human proteins. Hence, in biology
and biochemistry nanoparticles have attracted much
attention (Kildeby et al., 2006).
Green synthesis of nanoparticles
The concept of green chemistry has been appeared in
the United States, as a common research program
resulting from interdisciplinary co-operation of
university team, independent research groups, industry,
scientific societies and various governmental agencies,
which each have their own programs devoted to
decreasing pollution.
Green chemistry incorporates few new approaches to the
synthesis processing and application of chemical
substances in such a manner so as to reduce threats to
health and environment. These new approaches are
known as-
1) Environmentally Benign Chemistry
2) Clean Chemistry
3) Atom Economy
4) Benign-by-design Chemistry
The first principle describes the basic idea of green
chemistry; protecting the environment from pollution.
The remaining principles are focused on issues such as
atom economy, toxicity, solvent and other media using
consumption of energy, application of raw materials from
renewable sources and degradation of chemical products
to simple, nontoxic substance that are friendly for
environment. Green chemistry is an approach to design,
manufacture and use of chemical products to
intentionally reduce or eliminate chemical hazards.
Thus, goal of green chemistry is to create better, safer
chemicals while choosing the safest and most efficient
ways to synthesize them and reduce wastes (Wardencki
et al., 2005).
Benefits of green chemistry or green synthesis
 Economical
 Energy efficient
 Lower cost of production and regulation
 Lesser waste
 Fewer accidents
 Safer products
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 Competitive advantage
 Healthier work places and communities
 Protects human health, environment, compatible
for pharmaceutical and other biomedical
applications (Huang et al., 2004; Hebeish et al.,
2010; Park et al., 2011)
Need of green synthesis
A lot of interest has been created by the term "Green
Nanotechnology (GN)”. In a broad sense, this term
includes a wide range of possible applications from
nanotechnology enabled, environmentally friendly
manufacturing processes that reduce waste products
(ultimately leading to atomically precise molecular
manufacturing with zero waste); the use of
nanomaterials as catalysts for greater efficiency in
current manufacturing processes by minimizing or
eliminating the use of toxic materials (green chemistry
principle); the use of nanomaterials and nanodevices to
reduce pollution (e.g., water and air filters); and the use
of nanomaterials for more efficient alternative energy
production (e.g., solar and fuel cells) (Benjamin et al.,
2011).
Green synthesis of silver nanoparticles (Ag NPs)
Silver nanoparticles (Ag NPs) or nanosilver (NS) are
clusters of silver atoms that range in diameter from 1 to
100 nm and are attracting interest as antibacterial and
antimicrobial agents for applications in medicine. NS is a
blossoming field of research and has been highly
commercialized. Colloidal silver is of particular interest
because of distinctive properties, such as good
conductivity, chemical stability, catalytic and
antibacterial activity (Frattini et al., 2005). Chemical
reduction is the most frequently applied method for the
preparation of silver nanoparticles (Ag NPs) as stable,
colloidal dispersions in water or organic solvents (Wiley
et al., 2005; Tao et al., 2006). Commonly used reductants
are borohydride, citrate, ascorbate and elemental
hydrogen. The reduction of silver ions (Ag+) in aqueous
solution generally yields colloidal silver with particle
diameters of several nanometers (Wiley et al., 2005;
Merga et al., 2007).
The green synthesis of silver nanoparticles (Ag NPs)
involves three main steps, which must be evaluated
based on green chemistry perspectives, including-
1) Selection of solvent medium.
2) Selection of environmentally benign reducing
agent.
3) Selection of nontoxic substances for the Ag NPs
stability (Huang et al., 2004).
The applications of nanoscale materials and
structures, is presently an emerging area of nanoscience
and nanotechnology. Clothing manufacturers have
incorporated NS into fabrics for socks and exploit the
antibacterial activity for neutralization of odor-forming
bacteria. In addition, NS has been integrated into
various food contact materials such as plastics used to
fabricate food containers, refrigerator surfaces, storage
bags and chopping boards, under the pretext of
preserving foods longer by inhibiting microorganisms
2330
growth. Nanomaterials are providing solutions to many
technological and environmental challenges in the areas
of solar energy conversion, catalysis, medicine and water
treatment (Dahl et al., 2007; Hutchison 2008). This
increasing demand along with safety must be
accompanied by “green synthesis methods”. In the global
efforts to reduce generated hazardous waste, green
chemistry and chemical processes are progressively
integrating with modern developments in science and
industry (DeSimone, 2002; Gross et al., 2002).
Nanomaterials often show unique and considerably
changed physical, chemical and biological properties
compared to their macro scaled counterparts (Li et al.,
2001). Thus synthesis of noble metal nanoparticles for
ample of applications has become an area of constant
interest. Studies have shown that the size, morphology,
stability and properties (chemical and physical) of the
metal nanoparticles are strongly influenced by the
experimental conditions, the kinetics of interaction of
metal ions with reducing agents, and adsorption
processes of stabilizing agent with metal nanoparticles.
Hence, the design of a synthesis method in which the
size, morphology, stability and properties are controlled
has become a major field of interest (Masciangioli et al.,
2003; Burleson et al., 2005; Albrecht et al., 2006).
Methods for green synthesis of silver nanoparticles
1) Polysaccharide method
In this method, Ag NPs are prepared by using
water as an environmentally benign solvent and
polysaccharides as a capping agent, or in some
cases polysaccharides serve as both a reducing
and a capping agent. For instance, synthesis of
starch-Ag NPs is carried out with starch as a
capping agent and β-D-glucose as a reducing agent
in a gently heated system (Raveendran et al.,
2003). The starch in the solution mixture avoids
use of relatively toxic organic solvents (Amanullah
et al., 2005). Additionally, the binding interactions
between starch and Ag NPs are weak and can be
reversible at higher temperatures, allowing
separation of the synthesized particles (Figure 1).
In a case of dual polysaccharide function, Ag NPs
were synthesized by the reduction of Ag+ inside of
nanoscopic starch templates (Raveendran et al.,
2003 and 2005).
Also, Ag NPs are synthesized by using negatively
charged heparin as a reducing or stabilizing agent
by heating a solution of AgNO3 and heparin to
70 °C for 8 hr (Huang et al., 2004).
Stable Ag NPs (10–34 nm) can also be synthesized
by autoclaving a solution of AgNO3 and starch
(capping/reducing agent) at 15 psi and 121 °C for 5
min. (Vigneshwaran et al., 2006). The Ag NPs are
stable in solution for three months at 25 °C.
Smaller Ag NPs (≤10 nm) are produced by mixing
two solutions of AgNO3 containing starch, a
capping agent and NaOH solution containing
glucose a reducing agent, in a spinning disk
reactor with a reaction time of less than 10 min.
(Tai et al., 2008). Importantly, starch-protected
Int J Pharm Sci Nanotech Vol 7; Issue 1  JanuaryMarch 2014
nanoparticles can be easily integrated into
systems for biological and pharmaceutical
applications (Sharma et al., 2009).
Fig. 1. TEM image of starch silver nanoparticles synthesized by
polysaccharide method.
2) Irradiation method
Ag NPs can be successfully synthesized by using a
variety of irradiation methods. For example, laser
irradiation of an aqueous solution of Ag salt and
surfactant can fabricate Ag NPs with a well
defined shape and size distribution (Abid et al.,
2002). No reducing agent is required in this
method. Additionally, laser is applied in a photo-
sensitization technique for the synthesis of
Ag NPs using benzophenone. Here, low laser
powers at short irradiation times gives Ag NPs of
nearly 20 nm, while an increased irradiation
power gives nanoparticles of nearly 5 nm. The
formation of Ag NPs by this photo-sensitization
technique is also achieved by using a mercury
lamp (Eustis et al., 2005). In the visible light
irradiation studies, photo-sensitized growth of Ag
NPs using thiophene as a sensitizing dye and Ag
NPs production by illumination of Ag(NH3)+ in
ethanol has been carried out (Zhang et al., 2003).
Synthesis procedures using microwave irradiation
has also been employed. Microwave irradiation of
a carboxymethyl cellulose sodium and silver
nitrate solution produce uniform Ag NPs that are
stable for two months at room temperature (Chen
et al., 2008). Recently, the use of microwave
radiation to synthesize nearly mono-dispersed
Ag NPs using basic amino acids as reducing
agents and soluble starch as a protecting agent
has been shown (Hu et al., 2008; Sharma et al.,
2009).
3) Biological method
Extracts from bio-organisms may act both as
reducing and capping agent in Ag NPs synthesis.
The reduction of Ag+ ions by combinations of
biomolecules found in these extracts such as
enzymes, proteins, amino acids, polysaccharides
and vitamins is environmentally benign, yet
Bhosale et al: Innovative Eco-friendly Approaches for Green Synthesis of Silver Nanoparticles
chemically complex (Jagadeesh et al., 2004;
Collera et al., 2005).
For example, the extract of unicellular green algae
Chlorella vulgaris is used to synthesize single-
crystalline Ag nanoplates at room temperature.
Proteins in the extract provide dual functionality
of Ag+ reduction and shape-control in the NS
synthesis. The carboxyl groups in aspartic and/or
glutamine residues and the hydroxyl groups in
tyrosine residues of the proteins are suggested to
be responsible for the Ag+ ion reduction (Xie et al.,
2007).
Plant extracts from live alfalfa, the broths of
lemongrass, geranium leaves and others serve as
green reactants in Ag NPs synthesis (Gardea et al.,
2003; Shankar et al., 2003 and 2005). The reaction
of aqueous AgNO3 with an aqueous extract of
leaves of a common ornamental geranium plant
Pelargonium graveolens gives Ag NPs after 24 hr
(Shankar et al., 2003).
Several microorganisms are utilized to grow
Ag NPs intracellularly or extracellularly. For
instance, Ag containing nanocrystals of different
compositions are synthesized by Pseudomonas
stutzeri AG259 bacterium (Klaus et al., 1999). The
white rot fungus, Phanerochaete chrysosporium
and a marine fungus Penicillium fellutanum also
reduces Ag+ ion to form Ag NPs; where a protein is
suggested to cause the reduction (Vigneshwaran
et al., 2007) (Figure 2).
Possible involvement of proteins in synthesizing
Ag NPs is observed in filamentous-cyano-
bacterium Plectonema boryanum UTEX 485
(Lengke et al., 2007). Moreover, Ag+ reduction by
culture supernatants of colonic flora Klebsiella
pneumonia, Escherichia coli and Enterobacter
cloacae (Enterobacteriacae) produces rapid
formations of Ag NPs (Shahverdi et al., 2007).
Fig. 2. TEM micrograph of silver nanoparticles synthesized by
Penicillium fellutanum.
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4) Polyoxometalates method
Polyoxometalates (POMs) have the potential of
synthesizing Ag NPs because they are soluble in
water and have the capability of undergoing
stepwise multi-electron redox reactions without
disturbing their structure as shown in Figure 3
(Weinstock 1998; Hill 1998; Troupis et al., 2002).
In this method, POMs serve both as a photo
catalyst, a reducing agent and as a stabilizer. This
method does not use a catalyst or a selective
etching agent. Ag NPs of different shape and size
can be obtained using different POMs (Troupis
et al., 2002).
Fig. 3. TEM image of Ag NPs synthesized by POMs method.
5) Tollens method
The Tollens synthesis method gives Ag NPs with a
controlled size in a one-step process. The basic
Tollens reaction involves the reduction of
Ag(NH3)2+ (aqueous) a Tollens reagent, by an
aldehyde,
Ag(NH3)2+(aq) + RCHO(aq) → Ag(s) + RCOOH(aq)
In the modified Tollens procedure, Ag+ ions are
reduced by saccharides in the presence of
ammonia, yielding Ag NP films with particle sizes
from 50–200 nm, Ag hydrosols with particles in
the order of 20–50 nm and Ag NPs of different
shapes (Figure 4). Ag(NH3)2+ is a stable complex
ion resulting from ammonia's strong affinity for
Ag+, therefore the ammonia concentration and
nature of the reductant play a major role in
controlling the Ag NPs size (Saito et al., 2003;
Kvitek et al., 2005).
Silver nanoparticles incorporation into other
materials
The unique properties of Ag NPs have been extended
into a broader range of applications. Incorporation of
Ag NPs into other materials is an attractive method of
increasing compatibility for specific applications.
2332
Methods
 Silver-doped Hydroxyapatite.
 Silver-polymer Nanoparticles.
1) Silver-doped hydroxyapatite
There is great interest in inorganic-inorganic hybrid
nanocomposites materials because of their
industrial and medical applications. Recently, one-
step synthesis of anisotropic Ag nanocrystals is
achieved by reducing aqueous Ag+ ion by the
electron transfer from the surface of hydroxyapatite
(HA). The hydroxyl group in this process acted both
as a reducing and a binding agent to give highly
oriented flat rod and needle-like Ag NPs
(Arumugam et al., 2007). A microwave process is
also applied to synthesize nanosized Ag-substituted
HA with a length of 60–70 nm and width of 15-20
nm (Ramesh babu et al., 2007).
2) Silver-polymer nanoparticles
Nano composites consisting of metallic
nanoparticles incorporated in/with polymers have
attracted much attention because of their distinct
optical, electrical and catalytic properties which
have potential applications in the fields of catalysis,
bio-engineering, photonics and electronics (Kiesow
et al., 2003; Min et al., 2008). Polymers are
considered as a good host material for metal
nanoparticles as well as for other stabilizing agents
such as citrates, organic solvents, long chain
alcohols, surfactants and organometallics (Chen
et al., 2001; Sarkar et al., 2005). Different physical
and chemical methods exist to prepare
metalpolymer composites. A successful preparation
of NPs is determined by the ability to produce
Int J Pharm Sci Nanotech Vol 7; Issue 1  JanuaryMarch 2014
Fig. 4. TEM images of silver
nanoparticles- a) cubes; b)
triangles; c) wires; d) an
alignment of wire.
particles with uniform distributions and long
stability, given their tendency to rapidly
agglomerate in aqueous solution (Rifai et al., 2006;
Min et al., 2008). The main fabrication approach is
to disperse previously prepared particles in the
polymer matrix. This method is often referred to as
the evaporation method since the polymer solvent is
evaporated from the reaction mixture after NPs
dispersion (Zheng et al., 2001; Perkas et al., 2008).
E.g., Poly(vinyl alcohol)-silver nanoparticles
(Sharma et al., 2009).
Characterization of silver nanoparticles
1) Particle size and size distribution
The particle size is one of the most important
parameters of NPs. Two techniques are used to
determine the particle size distribution of NPs
which includes Photon Correlation Spectroscopy
(PCS) and Electron Microscopy (EM). The latter
includes Scanning Electron Microscopy (SEM),
Transmission Electron Microscopy (TEM) and
freeze-fracture techniques. The size evaluation of
nanoparticle dispersion demonstrates better
results, with freeze-fracturing microscopy and
photon correlation spectroscopy as quantitative
methods. Electron microscopy, however, could be
adopted as an alternative option that measures
individual particle for size and distribution. It is
relatively less time consuming. Additionally, the
freeze-fracturing of particles allows for
morphological determination of their inner
structure. In combination with freeze-fracture
procedures, TEM permits differentiation among
Bhosale et al: Innovative Eco-friendly Approaches for Green Synthesis of Silver Nanoparticles
nanocapsules, nanoparticles and emulsion
droplets. Atomic Force Microscopy (AFM) is an
advanced nanoscopic technique applied for the
characterization of NPs. AFM images can be
obtained in an aqueous medium, and for this
reason it is an effective means of investigating
NPs behavior in a biological environment.
Mercury porositometry is an equally suitable
technique for the sizing of NPs. The freeze-dried
NPs are filled in a dilatometer under vacuum and
then measured with the help of a mercury
pressure porositometer. This method largely
measures particulate agglomerates as mercury
fails to penetrate to a greater extent within the
primary particles. The dry powders of the silver
nanoparticles are also used for X-ray diffraction
(XRD) analysis, which is used to determine
crystalline particles. Here, crystalline size can be
determined by using formula as Scherrer’s
equation as follows-
T = 0.94λ/βcos
Where, T = crystallite size, λ = wavelength of the
radiation, θ = Bragg's angle and β = full width at
half maximum
2) Specific surface area
The specific surface area of freeze-dried NPs is
generally determined with the help of a
sorptometer. The equation given below can be
used to calculate specific surface area:
A = 6 / ρd
Where A is the specific surface area, ρ is the
density and d is the diameter of the particle.
In most cases, the measured and calculated
specific surface areas fairly comply while in some
cases, the residual surfactant could produce a
deviation in the measured values.
3) Surface charge and electrophoretic mobility
The nature and intensity of the surface charge of
NPs is very important as it determines their
interactions with the biological environment as
well as their electrostatic interaction with
bioactive compounds. The surface charge of
colloidal particles in general and NPs in particular
can be determined by measuring the particle
velocity in an electric field. Laser light scattering
techniques such as Laser Doppler Anemometry or
Velocimetry (LDA/LDV) are used as fast and high-
resolution techniques for determining NPs
velocities. The surface charge of colloidal particles
can also be measured as electrophoretic mobility.
The charge composition critically decides the bio-
distribution of drug carrying NPs. Generally, the
electrophoretic mobility of NP is determined in a
phosphate saline buffer and human serum. The
phosphate saline buffer (pH 7.4) reduces the
absolute charge value due to ionic interaction of
buffer components with the charged surface of
nanoparticles. The zeta potential can be obtained
by measuring the electrophoretic mobility by
applying the Helmholtz-Smoluchowski equation.
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4) Surface hydrophobicity
The surface hydrophobicity of NPs influences the
interaction of colloidal particles with the biological
environment. Hydrophobicity and hydrophilicity
collectively determine the bio-fate of NPs and
their contents. Hydrophobicity regulates the
extent and type of hydrophobic interactions of NPs
with blood components. Several methods including
hydrophobic interaction chromatography, two-
phase partition, adsorption of hydrophobic
fluorescent or radiolabelled probes and contact
angle measurements have been adopted to
evaluate surface hydrophobicity. Recently, several
sophisticated methods of surface chemistry
analysis have also been used.
5) Density
The density of NPs is determined with helium or
air using a gas pycnometer. The value obtained
with air and helium is much more pronounced due
to the specific surface area and porosity of the
structure.
6) Molecular weight measurements
The molecular weight of the polymer and its
distribution in the matrix can be evaluated by Gel
Permeation Chromatography (GPC) using a
refractive index detector. Using GPC, it was
shown that polyalkylcynoacrylate (PACA)
nanoparticles are built by an entanglement of
numerous small oligomeric subunits rather than
by the rolling up of one or a few long polymer
chains.
7) Nanoparticle recovery and drug incorporation
efficiency
Nanoparticle recovery also referred to as
nanoparticle yield, can be calculated using the
following equation:
Nanoparticle recovery (%) = Concentration of drug
in nanoparticles/Concentration of nanoparticles
recovered × 100.
Drug incorporation efficiency has been expressed
both as drug content (% w/w), also referred to as
drug loading and drug entrapment (%)
represented by the following equation:
Drug incorporation efficiency in nanoparticles =
Amount of drug entrapped in nanoparticles/Total
amount of added drug.
8) In-vitro release
The in vitro release profile of nanoparticles can be
determined using standard dialysis, diffusion cell
or modified ultra-filtration techniques which have
been recently introduced and which use phosphate
buffer utilizing double chamber diffusion cells on a
shake stand. A Millipore, hydrophilic, low protein-
binding membrane is placed between the two
chambers. The donor chamber is filled with NPs
suspension and the receptor compartment is
assayed at different time intervals for the released
drug using standard procedures. The modified
ultra-filtration technique is also used to determine
the in-vitro release behavior of NPs. Here the NPs
2334
suspension is added directly into a stirred ultra-
filtration cell containing buffer. At different time
intervals, aliquots of the dissolution medium are
filtered through the ultra-filtration membrane
using < 2 positive nitrogen pressure and assayed
for the released drug using standard procedures
(Shobha Rani R., 2008).
Purification of silver nanoparticles
Depending on the preparation method used, various
impurities can be found in the NPs suspensions. Simple
filtration will only remove polymer aggregates, while
other impurities require more sophisticated procedures.
The most commonly used procedures are gel filtration,
dialysis and ultracentrifugation. However, these methods
are not entirely satisfactory because they are restricted
to the lab scale or incapable of eliminating molecules
with high molecular weights. The necessity of finding an
efficient purification technique that can be scaled up
from an industrial standpoint led to the development of
the cross-flow filtration method. In this method, the NPs
suspension is filtered through membranes, with the
direction of the fluid being tangential to the surface of
the membranes. The suspension is submitted to several
filtration cycles while the filtrate containing components
smaller than the pores of the membrane as well as
soluble impurities is discarded. Most importantly, this
technique can be scaled up by enlarging the filtering
surface (Shobha Rani R., 2008).
Sterilization of silver nanoparticles
Silver nanoparticles intended to be used parenterally
are required to be sterile and non-pyrogenic. Some of the
well established methods of sterilization such as
filtration through 0.22 µm filters are not adequate for
nanoparticle suspensions because microorganisms and
nanoparticles are generally similar in size (0.3-1 µm).
Therefore, sterilization of silver nanoparticles is best
achieved by using aseptic techniques throughout their
processing and preparation. However, this is not a totally
fullproof method and therefore, a terminal sterilization
step is required to ensure the microbiological safety of
the final product. Autoclaving (moist heat sterilization)
and γ-radiation are the techniques that can be applied for
terminal sterilization (Shobha Rani R., 2008).
Applications of silver nanoparticles
Silver nanoparticles are being used in numerous
technologies and incorporated into a wide array of
consumer products that take advantage of their desirable
optical, conductive and antibacterial properties.
Catalytic actions: High surface area and high surface
energy predetermine metal nanoparticles for being
effective catalytic medium. Growing small particles of
silver have been observed to be more effective catalysts
than stable colloidal particles. These growing particles
catalyse the borohydride reduction of several organic
dyes. The reduction rate catalyzed by growing particles is
distinctly faster compared to that of stable and larger
silver particles, which are the final products of growing
particles. Catalysis is due to efficient particle-mediated
Int J Pharm Sci Nanotech Vol 7; Issue 1  JanuaryMarch 2014
electron transfer from the ion to dye, and the dye
particles interaction. Catalytic activity of silver
nanoparticles can be controlled by its size, as redox
potential depends on the nanoparticles size (Kholoud et al.,
2010).
Antimicrobial agent: Silver is a nontoxic, safe
inorganic antibacterial agent being used for centuries
and is capable of killing about 650 microorganisms that
cause diseases. Silver has been described as being
‘oligodynamic’, that is its ions are capable of causing a
bacteriostatic or even a bactericidal impact. It has the
ability to exert a bactericidal effect at minute
concentration. It has a significant potential for a wide
range of biological application such as antibacterial
agents for antibiotic resistant bacteria, preventing
infections, healing wounds and as anti-inflammatory.
Silver ions and its compounds are highly toxic to
microorganism exhibiting strong biocidal effect on many
species of bacteria but have a low toxicity towards
animal cells. Therefore, silver ions being antibacterial,
are employed in formulation of dental, resin composites,
bone cement, ion exchange fibers and coatings for
medical devices. Ag NPs also find applications in topical
ointments and creams used to prevent infection in burns
and open wounds. Anticancer particles with paclitaxel
inhibit the growth of hep G2 cell more effectively and
found to be nontoxic on live cells and induce apoptosis on
cancerous HT29 (Kholoud et al., 2010; Sadowski, 2010;
Kannan et al., 2011).
Cell imaging: Aptamer based silver nanoparticles are
used in intracellular protein imaging and single
nanoparticle spectral analysis, where Ag NPs act as an
illumiophore and the aptamer as a biomolecule specific
recognition unit (Rosarin and Mirunalini, 2011).
Biosensors: Prostate Specific Antigen (PSA) has been
used to confirm the presence of semen and absence of
sperm in sexual assault cases. The use of PSA in forensic
analysis has a different set of requirements such as lack
of sample or need to extract. Nanoparticles and
nanostructures are used to enable the analytic detection.
Ag NPs probes are attached to antibodies that are
labeled with Raman dyes and in this method PSA
detection is done by enhancement of Raman Scattering.
This assay has been regarded robust for PSA detection
due to being label free and inexpensive (Rosarin and
Mirunalini, 2011).
Cancer therapy (Anticancer Tool): Conventional
strategies for cancer intervention include surgery,
chemotherapy and radiation therapy. Taking advantage
of their unique properties, most studies of gold and Ag
NPs based cancer therapy have used photothermal
therapy for the destruction of cancer cells or tumor
tissue, which may be potentially useful in the clinical
setting (Rosarin and Mirunalini, 2011).
Anti-HIV tool: Recently, a study revealed the
potential cytoprotective activity of Ag NPs towards HIV-
1 infected cells. The activity of Ag NPs towards HIV-1
infected Hut/CCR5 cells was investigated using Terminal
Uridyl-Nucleotide End Labeling (TUNEL) assay after a
three day treatment. The percentage of aproprotic cells
Bhosale et al: Innovative Eco-friendly Approaches for Green Synthesis of Silver Nanoparticles
were determined as 49%, 35%, and 19% for vehicle
control, 5 μM Ag, and 50 μM Ag, respectively. Ag NPs
might inhibit the replication in Hut/CCR5 cells causing
HIV-associated apoptosis. Size dependent interaction of
Ag NPs with HIV-1 virus has also been demonstrated.
Ag NPs preferentially binds to gp120 glycoprotein knobs
of HIV-1 virus. In the In-vitro study, it was further
shown that this interaction caused the virus not to bind
with the host cell (Sharma et al., 2009).
Diagnosis: Gold nanoparticles as a class of
nanomaterials with many unique properties such as
colorimetric, conductivity and nonlinear optical
properties have been explored for potential applications
in biomolecular detection. NPs can be used to
quantitatively detect nucleic acids and proteins in
clinical samples. For example, a DNA based method for
rationally assembling gold nanoparticles into
macroscopic materials has been reported by Mirkin and
coworkers. For protein detection, an aggregation based
immunoassay for antiprotein A using gold NPs has been
developed. The hyper-Rayleigh scattering signals of
aggregated gold nanoparticles labeled with
immunoglobulin G could be used to quantify antibody or
antigen in aqueous solution (Rosarin and Mirunalini,
2011).
Antioxidant properties: Previously it was reported
that Ag NPs reduce mitochondrial function and increase
membrane leakage in mammalian germ-line stem cells,
increase Reaction Oxygen Species (ROS) generation,
deplete antioxidant GSH content and reduce
mitochondrial function in BRL 3A rat liver cells. In
addition to the well known specific effects of peroxide,
superoxide and hydroxyl radicals, ROS in general also
cause DNA damage including a multitude of oxidized
base lesions, abasic sites, single and double-strand
breaks; all of these can be cytotoxic and/or mutagenic.
Excessive ROS generation in cell is known to induce
apoptosis or cell death. ROS generation has been shown
to play an important role in apoptosis and can be treated
with Ag NPs. It has been studied that Ag NPs increase
the production of intracellular ROS and decrease reduced
glutathione (GSH) levels, leading to apoptosis. It shows
that Ag NPs decrease GSH levels through the inhibition
of GSH synthesizing enzymes (GCLS and GSS), leading
to the generation of ROS. However, GSH is not the only
intracellular antioxidant of defense; therefore it remains
to be determined whether other intracellular antioxidant
systems like superoxide dismutase and catalase are also
impaired by Ag NPs (Rosarin and Mirunalini, 2011).
Conductive applications: Ag NPs are used in
conductive inks and integrated into composites to
enhance thermal and electrical conductivity (Kannan et al.,
2011).
Optical applications: Ag NPs are used to efficiently
harvest light and for enhanced optical spectroscopy
including Metal-Enhanced Fluorescence (MEF) and
Surface-Enhanced Raman Scattering (SERS) (Hu et al.,
2008; Rosarin and Mirunalini, 2011).
2335
Summary and future perspectives
Nanotechnology is an emerging field that has made
its contribution to all spheres of human life. Green
synthesis of NPs is found to be an emerging branch of
nanotechnology that has paved for better methodologies
and approaches in the medicinal field. The use of
environmentally benign materials like plant extracts for
the synthesis of nanoparticles offers numerous benefits of
eco-friendliness and compatibility for pharmaceutical
and biomedical applications as they do not use toxic
chemicals in the synthesis protocols. Additionally, bio-
inspired synthesis of nanoparticles provides
advancement over chemical and physical methods as it is
a cost effective and environment friendly and in this
method there is no need to use high pressure, energy,
temperature and toxic chemicals.
Green synthetic methods using various plant extracts
have proven a great potential in Ag NPs synthesis, which
are widely used as novel therapeutic agents nowadays.
But still, further advancements in green synthesis,
purification and sterilization methods will take
nanotechnology to a new-fangled green path in near
future.
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Address correspondence to: Rohit Rajendra Bhosale, Department of
Pharmaceutics, Satara College of Pharmacy, Satara, Plot No. 1539, New
MIDC, Degaon, Satara-415 004, Maharashtra, India.
Tel.: 09579724707; E-mail: bhosalerohit707@gmail.com