Annals of Phytomedicine

Abstract

Mini-implants have become a major device in orthodontic treatment in this era, and practitioners intend to use for different clinical situations. Silver nanoparticle coating onto metal is known to reduce the colonization of bacteria, thus coating mini-implants will reduce the risk of peri-implantitis and implant failure. As there are new horizons being explored with nanotechnology, assessing its toxicity is very essential for its use in medicine. Chitosan based silver impregnated nanoparticles were synthesised using green tea extract and chitosan particles using thermal treatment. Formation of silver nanoparticles were assessed using double beam UV spectropho-tometer. Mini-implants coated with silver nanoparticles were tested for antimicrobial property against Strep-tococcus mutans, Staphylococcus aureus, Lactobacillus and Candida albicans. Characterisation of the surface coating was done by using TEM. To assess the cytotoxic potential of silver nanoparticles, shrimp was cultured and were tested by adding various concentration of silver nanoparticle to it. UV-vis spectrophotometer was used to examine the formation of silver nanoparticles at 440 a.u peak. Transmission electron microscope (TEM) was used to characterize the nanoparticles that showed particles spherical in shape. A considerable zone of inhibition was also formed after 24 h of incubation for bacteria and 48 h of incubation of silver nanoparticles in Candida albicans. Cytotoxicity was assessed by testing on shrimp culture. Titanium mini-implants when coated with silver nanoparticles has excellent antimicrobial properties and, hence can be used a biomaterial in orthodontics but further tests are needed to evaluate the coating during and after placement.

Full text

207
Article Info
Article history
Received 11 April 2020
Revised 25 May 2020
Accepted 27 May 2020
Published online 30 June 2020
Keywords
Silver nanoparticles
Chitosan
Mini-implant
Orthodontics
Antimicrobial
Cytotoxi ci ty
Original article: Open access
Assessment of antimicrobial activity and cytotoxic effect of green mediated silver
nanoparticles and its coating onto mini-implants
Swapna Sree nivasagan, Ar avind Kumar Subr amanian* and S. Rajeshkumar**
Department of Orthodontics, Saveetha Dental College, No. 162, Saveetha Institute of Medical and Technical Sciences, Saveetha University,
Chennai-6000 77, T.N., India
*Department of Orthodontics, Saveetha Dental College, No. 162, Saveetha Institute of Medical and Technical Sciences, Saveetha University,
Chennai-6000 77, T.N., India
**Department of Pharmacology, Saveetha Dental College, No. 162, Saveetha Institute of Medical and Technical Sciences, Saveetha University,
Chennai-6000 77, T.N., India
Abstract
Mini-implants have become a major device in orthodontic treatment in this era, and practitioners intend to use
for different clinical situations. Silver nanoparticle coating onto metal is known to reduce the colonization of
bacteria, thus coating mini-implants will reduce the risk of peri-implantitis and implant failure. As there are new
horizons being explored with nanotechnology, assessing its toxicity is very essential for its use in medicine.
Chitosan based silver impregnated nanoparticles were synthesised using green tea extract and chitosan particles
using thermal treatment. Formation of silver nanoparticles were assessed using double beam UV spectropho-
tometer. Mini-implants coated with silver nanoparticles were tested for antimicrobial property against Strep-
tococcus mutans, Staphylococcus aureus, Lactobacillus and Candida albicans. Characterisation of the surface
coating was done by using TEM. To assess the cytotoxic potential of silver nanoparticles, shrimp was cultured
and were tested by adding various concentration of silver nanoparticle to it. UV-vis spectrophotometer was used
to examine the formation of silver nanoparticles at 440 a.u peak. Transmission electron microscope (TEM)
was used to characterize the nanoparticles that showed particles spherical in shape. A considerable zone of
inhibition was also formed after 24 h of incubation for bacteria and 48 h of incubation of silver nanoparticles
in Candida albicans. Cytotoxicity was assessed by testing on shrimp culture. Titanium mini-implants when
coated with silver nanoparticles has excellent antimicrobial properties and, hence can be used a biomaterial in
orthodontics but further tests are needed to evaluate the coating during and after placement.
Copyright © 2020 Ukaaz Publications. All rights reserved.
Email: ukaaz@yahoo.com; Website: www.ukaazpublications.com
Annals of Phytomedicine 9(1): 207-212, 2020
Annals of Phytomedicine: An International Journal
http://www.ukaazpublications.com/publications/index.php
Print ISSN : 2278-9839 Online ISSN : 2393-9885
DOI: http://dx.doi.org/10.21276/ap.2020.9.1.27
Corresponding author: Dr. Swapna Sreenivasagan
Department of Ortho do nt ics, Saveetha Dental College, No 16 2,
Ponamallee High Road, Chennai-60007 7, T.N., India
E-mail: swapnasreenivasagan@gmail.com
Tel.: +91-9444406 70 4
1. Introduction
The use of mini-implants as an adjuvant in anchorage in orthodontic
treatment of malocclusion has now been widely accepted in the
present era. For anchorage control, various methods are used in
practice that includes the bracket placement, anchorage from
extraoral devices like headgear. The use of these type of anchorage
requires maximum patient compliance and care (Tseng et al., 2006).
Micro-screw mini-implants has been widely used because of its
anchorage potential, easy placement, removal and low cost. Many
factors influence the success of mini-implant like its placement,
type of bone, placement procedure, angulation and site. To ensure
success, it is important to prevent inflammation around the screw
implants (Park et al., 2006). In orthodontic patient, there is a
documented increase in Streptococcus and Lactobacillus (Pellegrini
et al., 2009). Inflammation is caused as this is a site of injury,
patient during initial days have poor maintenance of hygiene due to
pain in the site, this acts as a nidus for the growth of micro-organisms.
The use of nanoparticle coated material is now being accepted in
dentistry as various benefits have been demonstrated by certain
materials. In dentistry in general, silver nanoparticles are attempted
to be incorporated into a wide range of materials because of its
antimicrobial mechanism. Composites, adhesives, acrylic resin and
even intra-radicular medicaments in endodontics are all the various
materials that have previously been tried along with silver
nanoparticles (Borzabadi-Farahani et al., 2014). Most of the studies
are being conducted in an experiment to enhance the antimicrobial
and the anti-cariogenic potential of the various components of the
orthodontic materials (Borzabadi-Farahani et al., 2014). In
orthodontics there has been previous studies incorporating silver
nanoparticles in various materials like orthodontic, wires, adhesives,
composite, brackets, bands, module, mini-implants in order to
evaluate various properties like antibacterial effect, antiadherence,
reduce friction and so on. Silver nanoparticle has shown bactericidal
effect against gram negative micro-organisms. Silver is an unreactive
component and has low ionization potential and is, thus also stable
in aqueous solution, physiological fluids and solids. Silver
nanoparticles have previously been coated onto hexagon dental
implants and has shown positive antimicrobial activity against S.
aureus and Candida (Rajeshkumar and Bharath, 2017).

208
Chitosan poly (D-glucosamine) has been widely used for coagulation
of metal ion removal as chitosan easily forms chelate with cation
(Rajeshkumar and Bharath, 2017). Chitosan, a polysaccharide
biopolymer derived from naturally occurring chitin, displays unique
polycationic, chelating, and film-forming properties due to the presence
of active amino and hydroxyl functional groups (Agarwal et al., 2017).
A chitosan nanofiber scaffold can diminish infection in in vivo
implantation due to its antibacterial properties (Kohsari et al., 2016).
There are various methods for coating and deposition on the mini-
implants that have been used, the method ion include beam sputter
deposition the substrate is bombarded by energetic ions beam
becoming from inside an ion source; plasma ion etching method
(Zegan et al., 2017). There is also coating done with high pressure
Hg lamp in order for coating with various required concentrations
(Zhao et al., 2009). Various methods are available for characterization
of the nanoparticle and can be done using scanning electron
microscope (SEM), transmission electron microscope (TEM) and
atomic force microscope (AFM).
Studies have shown that from exposure to the oral cavity to both
ionic and nanoparticle silver, there has been deposition in the oral
epidermis, the glomeruli and in the intestines (Chang et al., 2006).
There is also evidence in literature that there is a continuous release
of silver ions from both silver nanoparticles and metallic silver
(Kittler et al., 2010). The lower ionic strength medium of silver
nanoparticles results in greater toxicity (Rajeshkumar and
Malarkodi, 2014). Silver when administered through oral route is
known absorbed in a range of 0.4-18% in mammalians and in man,
a value of 18% absorption. Silver is distributed to all of the organs
and highest levels are observed in the intestine and stomach. Silver
toxicity in skin is expressed as argyria. Excretion of silver is via
secretions of the bile and urine.
In the present age of technology there is wide range of studies that
are undertaken to study the antimicrobial property of nanoparticles
that are coated onto various materials that are used in the oral
environment. It is evident that there is some antibacterial property
in comparison to uncoated materials. The main objective of the
study is to evaluate the peak of formation of silver nanoparticles
under UV-vis spectrophotometer, to study the antibacterial activity
against Streptococcus mutans, Lactobacillus and Staphylococcus
aureus as these are the main bacteria found in increased levels in
orthodontic patients and antifungal activity against Candida
albicans. In this study we aim at synthesizing green mediated silver
nanoparticles, coating it onto mini-implants, test for antimicrobial
activity and to find the toxic potential of silver nanoparticles by
placing them in shrimp culture at their naupulii stage.
2. Materials and Methods
2.1 Synthesis of chitosan silver nanoparticles
0.5 g chitosan and 0.5 ml of acetic acid was measured and added to
49.5 ml of distilled water and allowed to mix using magnetic stirrer.
10 mM of silver nitrate in 10 ml of distilled water and these solutions
were mixed by continuous stirring to promote dissolution. Green
tea extract was prepared using boiling water. The extract solution
of the plant solution was added to the aqueous silver nitrate solution
and covered with aluminium foil and was subjected to magnetic
stirring for the next 8 h. All the containers were washed and rinsed
with deionized water prior to use. The mixture was separated into
test tubes and centrifuged at 8000 rpm for 15 min.
2.2 Coating of titanium mini-implants
Titanium mini-implants along with the solution containing silver
nanoparticles was placed in a magnetic stirrer followed by heating
and again stirring then, allow for the deposition of the particles on
the mini-implants.
2.3 UV vis spectroscopy and TEM analysis
Formation of silver nanoparticles by reduction of AgNO3 in the
chitosan solution was scanned between 250-700 a.u for every 1 h
for the next 6 h. The results were consistent with the typical spectra
for this solution. The absorption peaks that are formed between
390 and 470 a.u are of those related to different clusters of silver
ions (dos Santos et al., 2004). The surface morphology was assessed
using TEM.
2.4 Antimicrobial activity
Antimicrobial activity of silver nanoparticles was performed by
using agar well diffusion method. The muller Hinton agar for
antibacterial and rose Bengal agar for antifungal (Candida albicans)
was used. In order for the assessment of antimicrobial property
was assessed after the silver nanoparticles were formed by placing
50, 100 and 150 µl onto well cut in 4 petridishes and to test
an timicrobi al pr operty agai nst St reptoc occ ous mutan s,
Staphylococcus aureus, Lactobacillus and Candida albicans. The
result of antibacterial efficacy was examined after 24 h and
antifungal results after 48 h. After coating was completed, the coated
mini-implants were examined comparing with uncoated implants
to evaluate the antimicrobial efficacy against the same organism.
2.5 Toxic testing
Shrimp eggs were cultured using a culture containing iodine free
salt in water and sodium bicarbonate added to this as a source of
nutrition. Nauplii of the shrimp that were formed in 24 h were
collected and added 10 in one well to a 6 well ELISA plate. Silver
nanoparticles in varying concentrations of 5,10,15,20 and 25 µl
were added to each well and one well was used as a control. The
survival of the nauplii was assessed after 24 h.
3. Results
3.1 Antimicrobial activity results
The antibacterial activity of the synthesized silver nanoparticles
was co nfirmed by the zone of inhibi tion formed ag ainst
Staphylococcus aureus, Lactobacillus sp., Streptococcus mutans
as shown Table 1, Figure 1 and Figure 7. Strong antibacterial activity
was observed against Staphylococcus au reus, Lactobacillus,
antibacterial activity was observed against Streptococcus mutans
but the zone of inhibition was lesser than that observed in the other
bacteria. The synthesized nanoparticles did not have a very strong
antifungal activity.

209
Table 2, Figure 2 and Figure 8 showed the zone of inhibition around
the same micro-organisms in coated and uncoated mini-implants.
The zone of inhibition was measured as length by breadth
determined by the dimension of the mini-implants. There was no
bacterial growth around the coated mini-implants, there was no
inhibition of bacterial growth seen in the uncoated mini-implants.
Maximum zone of inhibition was observed for Staphylococcus
aureus and the least for Candida albicans.
Table 3 and Figure 3 showed the results of cytotoxicity tested and
the death and inhibition of growth of the organisms was observed
only in higher concentrations of the 20 and 25 µl. There was no
cytotoxic potential observed in lower concentrations.
3.2 Properties of silver nanoparticles
Figure 4 depicts the visual observation of the addition of green tea
extract, chitosan and silver nitrate, the solution initially was formed
in light brown colour and later on continued magnetic stirring changed
to dark brown colour. The results of UV vis spectroscopy is
depicted in the graph in Figure 5 where the peak for silver
nanoparticle formation is observed at a range of 250-700 nm. The
silver nanoparticles and silver nanoparticles impregnated chitosan
showed peak between 420-460 nm, confirms the silver nanoparticles
synthesis. The characterisation of the surface morphology of the
silver nanoparticles formed was studied under Transmission electron
microscope as observed in Figure 6 which revealed that the particle
size was formed of 30-35 nm and the particles were uniformly
distributed and spherical, triangular and hexagonal in shape.
Table 1: Zone of inhibition by silver nanoparticles
Zone of Inhibition (In mm)
Ba ct eria/ Fu ngi 50 µl 100 µl 150 µl
Staphylococcus aureus 9 18 20
Lactoba cillus 3 0 32 40
Streptococcus mutans 14 15 20
Candida albicans 9 10 12
Table 2: Length and breadth of zone of inhibition comparing silver
nanoparticle coated with uncoated mini-implant (Control)
Zone of inhibition formed (In mm)
Mi cro-o rg anism Coated Mini-implant Control
Len g t h Bre adt h Leng t h Br eadt h
Staphylococcus aureus 11 8 Nil Nil
Lactobacillus sp. 1 5 11 Nil Nil
Streptococcus mutans 15 10 Nil Nil
Candida albicans 12 6 Nil Nil
Table 3: Cytotoxicity: Assessing the number of live napulii after 24 h
Concentrat io n of silver No. of live orga nisms
nano pa rt ic le (In microlitre s)
5 10
10 10
15 10
20 8
25 5 Figure 4: UV-vis spectroscopic analysis.
Figure 1: Zone of inhibition by silver nanoparticles.
Zone of inhibition by silver nanoparticles
Figure 3: Assessing the number of live napulii after 24 h.
Figure 2: Length and breadth of zone of inhibition comparing silver
nanoparticle coated with uncoated mini-implant (Control).
Zone of inhibition comparing silver nanoparticles coated
with uncoated mini-implant

210
Figure 5: Visual observation.
Figure 8: Zone of inhibition of mini-implants coated with nanoparticles.Figure 7: Antimicrobial activity of silver participles.
Antimicrobial activity of silver nanoparticles
Figure 6: Transmission electron microscopic image of silver participles.

211
4. Discussion
Mini-implants are used for orthodontic anchorage and are usually
loaded early to reduce treatment time and is removed after treatment.
Mini-implants are placed near tooth roots (Park et al., 2006). The
principle factor for stability is only by mechanical lock to the bone.
Poor attention to oral hygiene lead to inflammation in the tissues
around the mini-implants and hastened their loss (Tseng et al., 2006).
In this study, the silver nanoparticles that were formed, showed a
peak of 450 nm. The silver nanoparticles had good antibacterial
effect even at low concentrations that increased as the concentration
was increased. Antifungal activity as present that was better at
higher concentration. TEM resu lts showed th at the silver
nanoparticles formed were of spherical, triangular and hexagonal in
shape and of the size of 30-35 nm. The toxicity results showed that
in lower concentrations all the nauplii survived, mild toxicity was
seen in higher concentrations.
Silver nanoparticles has properties such as optical and catalytic
properties, which depend on the size and shape of the produced
nanoparticles (Khodashenas and Ghorbani, 2019). Silver nanoparticles
have an antibacterial effect due to its activity of forming pits in the
cell wall of the bacteria. There is an evident increase in the cell
permeability that will result in cell death (Chaloupka et al., 2010).
A chitosan nanofiber scaffold can diminish infection in in vivo
implantation due to its anti-bacterial properties. Chitosan particles
gives a positive charge to the nanoparticles, this will thus increase
their binding to the negative charge of the bacterial cell wall
(Hernández-Sierra et al., 2008). Chitosan films with silver
nanoparticles antibacterial property against Escherisia coli for a
longer time than pure chitosan films (Agarwal et al., 2017). The
electrical and antibacterial properties of CS/AgNP and CS/Ag+ ions
bio nanocomposite depend on the concentration. Nanoparticles
research has reached unique interest because of its properties from
the bulk. Silver nanoparticles (Ag NPs) have gained substantial
attention due to their potential applications in medical field
especially in the production of biodegradable surgical sutures
(Kohsari et al., 2016). Silver particles have the various reactions
with a bacterial cell, that are interaction with cell wall causing lysis,
preventing DNA replication and also disrupts bacterial protein
synthesis (Chaloupka et al., 2010). Considering the liquid dilution
method to find the minimum inhibition concentration of 25 nm
AgNP is present against S. mutans and average of 4.8 µg/mL is also
responsible for the excellent antimicrobial activity of silver
nanoparticles (Hernández-Sierra et al., 2008).
Orthodontic wires coated with nickel-phosphorus film has shown
to reduce the friction which is a major factor that restricts treatment
(Redlich et al., 2008). Silver nanoparticle has been previously added
to adhesive used for bracket placement and has demonstrated
antimicrobial activity against S.mutans (Rajeshkumar and Bharath,
2017). In an in vitr o study when silver nanoparticles was
incorporated into the orthodontic band cement and antibacterial
activity was present for 28 days (Moreira et al., 2014). Various
nanoparticles have been coated onto orthodontic adhesive including
silver, zinc oxide and titanium oxide. Silver nanoparticles were coated
on modules and exhibited increased strength than conventional
modules (Hernández-Gómora et al., 2017). Titanium nanoparticle
incorporation in composite revealed lowering the colonisation of
S. mutans, S. sanguis and L. acidophilus and the shear bond strength
of the composite was reduced by still under acceptable levels
(Sodagar et al., 2016). In the field of implantology, nanoparticles
are deposited onto titanium mini-implants and has shown to be
advantageous for both cell proliferation and to prevent infection
from micro-organisms. Thermal annealing method in air had been
used to produce a thin film which was stable even in an aqueous
environment (Zegan et al., 2017). Bacterial adhesion onto titanium
implant is one of the major factor, hence surface modification by
coating with metallic nanoparticles will cause a decrease in the
colony forming units of bacteria and will be more beneficial for
treatment (García Contreras et al., 2011). Silver nanoparticles coated
onto orthodontic wires and brackets showed anti-adherence
properties of strains of Streptococcus mutans even in the presence
of orthodontic appliances (Espinosa-Cristóbal et al., 2018). As
white spot lesions are a known side effect to orthodontic treatment,
silver nanoparticles were also attempted to be used along with
orthodontic composite. Transbond as significant antibacterial
activity was demonstrated along against S. mutans (Sodagar et al.,
2016). On coating titanium mini-implants with biopolymer silver
nanoparticles, significant antimicrobial property has been
de mon str ate d a gainst S. muta ns, S. sang uin is, and A .
actinomycetemcometans. In situ release and good biocompatibility
was achieved in a previous study when silver nanoparticles were
incorporated into band cement that also had good antimicrobial
activity (Moreira et al., 2014).
Silver nitrate when at concentrations of 24 mM (corresponding to
308 mg of silver/kg of body weight) when present in the drinking
water can induce death over few days (Salhab et al., 1998). The
effects of particulate silver are mediated via silver ions releasing
from the particle surface (Hadrup and Lam et al., 2014). The potential
sources of AgNPs includes leaching of intact particles from consumer
products, disposal of waste from industrial processes, intentional
release into contaminated waters, and the natural formation of AgNPs
in surface and ground water (Sharma et al., 2019). The aggregation
of nanoparticle have an effect on toxicity by reducing the dissolution
rate and uptake by organisms and also the stability of these
nanoparticles (Yang et al., 2012). The colloidal spherical silver
nanoparticles and nano prisms are not genotoxic whereas silver
nitrate is genotoxic even at 10 µg/ml. Oxidative dissolution in
experimental conditions (maximally 15% in 24 h) is the key to the
toxicity of most Ag NPs, highlights a critical role for dissolved
silver complexed with thiols in the toxicity of all tested Ag NPs
(Yang et al., 2012).
5. Conclusion
Chitosan-silver nanoparticles have very strong antibacterial activity
against Staphylococcus aureus, Lactobacillus, antibacterial activity
was observed against Streptococcus mutans was minimal and the
synthesised nanoparticles did not have strong antifungal activity.
The precipitate shows peak between 420-460 nm, confirming the
silver nanoparticles synthesis. Further, studies are needed to
evaluate the long-term stability of the nanoparticle coating, the
integrity of the mechanical property of the materials coated and the
probable clinical use to reduce bacterial adherence and peri-
implantitis that are caused by oral micro-organisms. A limitation of
this study is that its results are only in vitro and toxicity needs in
to be checked in higher living organisms.

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Acknowledgements
We sincerely thank Indian Orthodontic Society Research Fund - Dr. C.
Padmalatha Memorial Grant for Research (Student Members) for
funding this study. We sincerely thank favanchor company for providing
their mini-implants for purpose of this study. We also sincerely thank
Ms M.Tarani for helping with the laboratory procedures.
Conflict of interest
The authors declare that there are no conflicts of interest in the course
of conducting the research. All the authors had final decision regarding
the manuscript and decision to submit the findings for publication.
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Citation: Swapna Sreenivasagan, Aravind Kumar Subramanian and S. Rajeshkumar (2020). Assessment of antimicrobial effects
and cytotoxic effect of green mediated silver nanoparticles and its coating onto mini-implants. Ann. Phytomed., 9(1):207-212.
http://dx.doi.org/10.21276/ap.2020.9.1.27