Development Of Anti Inflammatory and Antimicrobial Silver Nanoparticles Coated Suture Materials
Madhulaxmi1*, Jembulingam Sabarathinam2, Rajeshkumar3
1 Professor Department of Oral & Maxillofacial Surgery Saveetha Dental College & Hospitals Saveetha Institute Of Medical & Technical Sciences
Saveetha University, Chennai 600077, India.
2 Saveetha Dental College & Hospitals Saveetha Institute Of Medical & Technical Sciences (SIMATS) Saveetha University, Chennai 600077, India.
3 Associate Professor Department of Pharmacology, Saveetha Dental College and Hospitals, Saveetha Institute of Medical and Technical Sciences,
Saveetha University, Chennai-600077, Tamil Nadu, India.
*Corresponding Author
Madhulaxmi,
Professor Department of Oral & Maxillofacial Surgery Saveetha Dental College & Hospitals Saveetha Institute Of Medical & Technical Sciences Saveetha University, Chennai
600077, India.
Tel: 7373814000
E-mail: madhulaxmi@saveetha.com
Received: February 08, 2021; Accepted: March 03, 2021; Published: March 17, 2021
Citation: Madhulaxmi, Jembulingam Sabarathinam, Rajeshkumar. Development Of Anti Inflammatory and Antimicrobial Silver Nanoparticles Coated Suture Materials. Int J Dentistry
Oral Sci. 2021;08(03):2006-2013. doi: dx.doi.org/10.19070/2377-8075-21000395
Copyright: Madhulaxmi©2021. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution
and reproduction in any medium, provided the original author and source are credited.
Abstract
Background: Sutures are biomechanical devices used in oral surgery to avoid or limit the exposure of those parts exposed
to saliva from bacterial colonization. Nanotechnology witnessed tremendous growth and became an integral part of medical
sciences because of its wide range of application in different principles of diagnosis and treatment of various diseases. There
has been an expeditious advance in medicines and biomaterial in the recent years. Nanomedicine aids in prevention, diagnosis,
monitoring and treatment of the diseases.
Aim: The aim of the current study was to synthesize biogenic silver nanoparticles using Solanum nigrumand Indigofera
tinctoria as reducing agents and assess the antimicrobial, anti-inflammatory and the cytotoxic properties of the biogenic silver
nanoparticles. Further the study also aims to analyse the application of biogenic silver nanoparticles coated on surgical sutures.
Materials and Methods: In this report, we used the extract of Solanum nigrum and Indigofera tinctoria to synthesize silver
nanoparticles and were characterized using UV-visible spectrophotometer, SEM and TEM. These nanoparticles were subjected
to anti-inflammatory, Anti-microbial and and cytotoxic assays. Further these nanoparticles synthesized were coated on
surgical silk sutures and subjected to antimicrobial assay.
Results: The formed nanoparticles inherited important antibacterial properties against Pseudomonas sp, streptococcus mutan
and staphylococcus aureus. They also possess anti inflammatory activity and also prove to be biocompatible.
Conclusion: This break through in the field of surgical biomaterial may help reduce the toxic effects of pathogenic organisms
while representing an environmentally friendly material which would not only be economically efficient but also proves to be
a material with minimal risk to mankind and the environment.
2.Introduction
3.Materials and Methods
4.Results
5.Discussion
6.Conclusion
7.References
Keywords
Dentistry; Antimicrobial; Suture Materials; Anti-Inflammatory; Biocompatible; Silk.
Introduction
In the past decade, nanotechnology witnessed tremendous
growth and became an integral part of medical sciences because
of its wide range of application in different principles of diagnosis
and treatment of various diseases [1]. There has been an
expeditious advance in medicines and biomaterial in the recent
years [2]. Nanomedicine aids in prevention, diagnosis, monitoring
and treatment of the diseases. Silver Nanoparticles have always
been the nanoparticle of choice in modern medicine due their
significantly proven antimicrobial activity. Henceforth they play a
critical role in the development of modern science, especially in
the biological systems [3-8].
It is a well established fact that the size and shape determines the
properties of the silver nanoparticles. Moreover, their controllable synthesis proves to be a key challenge to acquire more desirable
characteristics [9]. Traditionally synthesized nanoparticles have
adverse side effects upon administration due to the highly toxic
chemicals which are being used for their synthesis [10]. Hence,
an alternative method of synthesis is required to overcome these
adverse effects of the nanoparticles.
Green chemistry suggests that there is a need for the majority
of the industries to become more sustainable through developing
eco-friendly products [11, 12]. Green chemistry offers alternative
approaches for synthesis of nanoparticles, by incorporating plant
extracts as they can be used to generate biogenic nanoparticles
[13, 14].
Solanum nigrum belongs to the solanaceae family, commonly
addressed as the makoi or blacknight shade. It is referred to as
Manathakkali in Tamil. S.Nigrum has been used for treatment of
fever, inflammation and stomach ulcers in Ayurveda (Traditional
medicine of India) [15]. It exhibits medicinal properties such as
antimicrobial activity, antioxidant activity, cytotoxic properties,
diuretic property, antiulcerogenic and hepatoprotective properties.
It is reported to be an alternative potential anti-cancer drug
[16, 17].
Indigofera tinctoria belongs to the family of Indirubins (bis-indoles),
commonly addressed as indigo plants. It is referred to as
avuri in Tamil. This plant is commonly cultivated in India and
china. This herb is widely used in indian system of medicine for
epilepsy, nervous disorder, bronchitis, and liver ailments.- Few literature
studies also reported that the herb can be used as an anticardiovascular
agent [18, 19].
Suture are biomechanical devices used in oral surgery to avoid or
limit the exposure of those parts exposed to saliva from bacterial
colonization [20]. Regardless, they offer adhesion to bacteria
on their surface which increases the susceptibility to postoperative
infections. Moreover surgical knots are reported to be the
fundamental site of bacterial colonization [21, 22]. Silk sutures
have been reported as the most commonly used suture material
for wound closure with acceptable results. However due to the
natural fibers being used in production of the silk sutures increase
the risk of development of infections [23]. Once the sutures are
infected, the local mechanisms are inactivated while some pathogens
are antibiotic resistant. Hence, tremendous efforts had been
taken to add antiseptics such as chlorhexidine and triclosan to the
sutures [24, 25]. Though few cases have been reported to be allergic
to these chemical substances [26].
Henceforth, the current study aims to synthesize biogenic silver
nanoparticles using Solanum nigrum and Indigofera tinctoria as
reducing agents and assess the antimicrobial, anti-inflammatory
and the cytotoxic properties of the biogenic silver nanoparticles.
Further the study also aims to analyse the application of biogenic
silver nanoparticles coated on surgical sutures.
Materials and Method
The herbs Solanum nigrum and Indigofera tinctoria were obtained
from green chem herbal extracts, Bangalore, India. 0.5 g of
dried S. nigrum and 0.5 g of dried I.tinctoria powder were transferred
into 100 mL of double distilled water in a 250 ml erlenmeyer
flask and boiled the mixture for 15 minutes. This process
extracted the components of the extracts in to water. Whatmann
No 1 filter paper was used for the filtration of boiling material,
to remove any suspended particles. Finally, a clear filtrate was obtained
and used for metal nanoparticle synthesis.
Biosynthesis Of Silver Nanoparticles (AgNPs) Using Leaf
Extract 1 mM Of AgNO3
The aqueous extract of S. nigrum and I. tinctoria was used for the
bioreduction process. For the biosynthesis of AgNPs, 20 mL of
aqueous extract was carefully added to 80 mL of 1 mM of aqueous
AgNO3 solution in 250 mL Erlenmeyer flasks. The flasks
containing the extract were incubated in an orbital shaker (FIGURE
1). The color changes in the reaction mixture was observed
continuously at various periods of time by double beam UV-vis
spectrophotometer at different wavelength regions from 350 to
550 nm. The biosynthesized silver nanoparticles were centrifuged
at 8000 rpm for 10 min and nanoparticles were collected.
Figure 1. Figure represents the reaction mixture placed on an orbital shaker in left and sedimentation on the biogenic nanoparticles in a cuvette on the right.
Characterization Of Prepared Nanoparticles
The synthesized nanoparticles were optically measured using double beam UV -vis spectrophotometer at different wavelength ranges of 350 to 550 nm. Morphological characterization of prepared nanoparticles was done using Transmission Electron Microscope (TEM) and Scanning electron microscope(SEM).
Antimicrobial Activity
The silver nanoparticles synthesized using S.nigrum and I.tinctoria were tested for antimicrobial activity by agar well diffusion method against surgical site pathogens namely S. mutans, S.aureus and Pseudomonas sp. The pure culture of these bacteria was subcultured on Mueller Hinton agar. Wells of 9 mm diameter were made on Muller Hinton agar plates using gel puncture and each strain was uniformly swabbed on to the individual plates using sterile cotton swabs. Using a sterile micropipette, a standard antibacterial agent was loaded into one well and three different concentrations of silver nanoparticles sample solutions (25 µl, 50 µl and 100 µl) were added onto three other wells on all plates. After incubation at 37°C for 24 hours, the different levels of zone of inhibition of bacteria were observed and measured.
Cytotoxicity Essay
Brine shrimp eggs were obtained from the Aquatic Remedies, Chennai. In this test, 6 elisa wells were filled with 2 g of non iodised salt dissolved in 200mL of distilled water. In each well, 10 nauplii ( freshly prepared brine shrimp larvae) were transferred. Among the 6 wells, 5 wells were loaded with AgNPs in different concentrations (10 microL, 20 microL, 30 microL, 40 microL, 50 microL). The last well was used as control without any nanoparticle. Finally the wells were filled with salt water till the brim and observed for changes after 24hrs (FIGURE 2).
Figure 2. ELISA wells with the nauplii.
Anti-Inflammatory Activity
Inhibition of Protein Denaturation method (MIZUSHTMA: 1966)
Concentration Of Test Substance: 10 – 50 µg/ml
Standard: Diclofenac sodium.
Chemicals Required: Bovine serum albumin, 1NHCl, Phosphate buffer (pH 6.3).
Instrument: Incubator, Spectrophotometer - 660nm.
The following 4 solutions were used
1. Test Solution (0.5ml) consist of 0.45ml of bovine serum albumin (5%w/v aqueous solution) and 0.05ml of test solution in various concentration and pH will be adjusted to 6.3 by using a small amount of 1N Hcl .The samples were incubated at 370C for 20 minutes and heated at 570C for 3 minutes(FIGURE 3). After cooling, to the sample add 2.5ml of Phosphate buffer (pH 6.3).
Figure 3. Incubation of the test and standard solutions after heating.
2. Test Control solution (0.5ml) consists of 0.45ml of Bovine serum albumin (5% aqueous solution) and 0.05ml of distilled water and pH will be adjusted to 6.3 by using a small amount of 1N Hcl the samples were incubated at 370C for 20 minutes and heated at 570C for 3 minutes. After cooling, to the sample add 2.5ml of phosphate buffer (pH 6.3).
3. Product Control (0.5ml) consists of 0.45ml of distilled water and 0.05ml of test solution in various concentrations and pH will be adjusted to 6.3 by using a small amount of 1N Hcl. The samples were incubated at 370C for 20 minutes and heated at 570C for 3 minutes. After cooling to the sample add 2.5ml to phosphate buffer (pH6.3).
4. Standard Solution (0.5ml) consists of 0.45ml of bovine serum albumin (5% w/v aqueous solution and 0.05ml of diclofenac sodium solution in various concentrations and pH will be adjusted to 6.3 by using a small amount of 1N Hcl. The samples were incubated at 370C for 20 minutes and heated at 570C for 3 minutes. After cooling, to the sample add 2.5 ml of phosphate buffer (pH6.3).
The percentage inhibition of Protein denaturation was calculated as follows.
OD of test solution - OD of product control
Percent Inhibition = 100 - -------------------------------------- X 100
OD of test control
The control represents 100% protein denaturation. The result will be compared with diclofenac sodium treated samples.
Antimicrobial Efficiency Of The AgNP Coated Sutures:
The silver nanoparticles synthesized using S. Nigrum and I. Tinctoria were coated on sutures by dipping the sutures in the nanoparticle solution and incubating for 24hrs. Then the coated sutures were tested for antimicrobial activity by agar well diffusion method against surgical site pathogens namely S. Mutans, S. Aureus and Pseudomonas sp sp. The pure culture of these bacteria was subcultured on Mueller Hinton agar. Each strain was uniformly swabbed onto the individual plates using sterile cotton swabs and the sutures were embedded. After incubation at 37°C for 24 hours, the different levels of zone of inhibition of bacteria were observed and measured.
Results Figure 4. Colour change of the reaction over the period of incubation time of 48hours. Figure 5. Graph of absorbance acquired from the UV vis spectrophotometry. The x axis represents the wavelength of while
the Y axis represents the absorbance. Figure 6. Biogenic silver nanoparticles under transmission electron microscope (TEM analysis). Figure 7. Biogenic silver nanoparticles under Scanning electron microscope (TEM analysis). Figure 8. Bar graph depicts the anti-inflammatory activity of biogenic silver nanoparticle; X axis represent the concentrations
of samples; Y axis denotes the inhibition percentage; red colour denotes the silver nanoparticles and yellow colour
represents the standard antiinflammatory agent (Control). Figure 9. Antimicrobial activity of biogenic silver nanoparticles by agar well diffusion method against surgical site pathogens
namely S. mutans, S.aureus and Pseudomonas sp. Figure 10. Bar graph depicts the measurement of zone of inhibition of biogenic silver nanoparticle; Y axis represent the
measurement of zone of inhibition; X axis denotes the specific organisms; Figure 11. Bar graph depicts the cytotoxic activity of Biogenic silver nanoparticles; Y axis represent the frequency of nauplii
alive; X axis denotes the concentration of nanoparticles. Figure 12. Antimicrobial activity of biogenic silver nanoparticle coated silk suture by agar well diffusion method against
surgical site pathogens namely S. Mutans, S.Aureus and Pseudomonas species. Figure 13. Bar graph depicts the measurement of zone of inhibition of biogenic silver nanoparticle coated silk sutures; Y
axis represent the measurement of zone of inhibition; X axis denotes the specific organisms;
Visual Analysis:
The present study reveals about the silver nanoparticles synthesized
using the herbs Solanum nigrum and Indigofera tinctoria
as bioreducing agents. The colour change in the reaction mixture
was observed continuously at various periods of incubation time.
The silver nitrate was reduced to silver nanoparticles, which was
visually indicated by colour change. The colour of the solution
changed from little green to dark brown in 24hours. The intensity
of the colour change gradually increased during the incubation
period. There was no colour change after 48 hours indicating that
the silver nanoparticle synthesis was completed.(FIGURE 4).
UV vis Spectrophotometer Analysis:
UV vis spectroscopy analysis depends on the arising of colour in
the reaction, due to excitation of surface plasmon resonance band
in the reaction mixture between different wavelengths from 350
to 550nm and was recorded. Figure 2 shows the UV absorbance
spectra of the synthesized silver nanoparticles using the extract
of the herbs Solanum nigrumand Indigofera tinctoria.
The figure depicts a steady increase in absorbance over the incubation
time period. The plasmon resonance band was peak in
365nm (FIGURE 5).
Transmission Electron Microscopy:
The structure of the AgNPs synthesized using the herbs Solanum
nigrum and Indigofera tinctoria as bioreducting agents are mostly
spherical and hexagonal in shape. The morphology of the silver
nanoparticles were highly variable with size range of 10-35nm
(FIGURE 6).
Scanning Electron Microscopy:
SEM analysis of the structure of the AgNPs synthesized using
the herbs Solanum nigrum and Indigofera tinctoria as bioreducting
agents are mostly spherical and elliptical in shape (FIGURE
7).
Anti-Inflammatory Activity:
The biogenic silver nanoparticles show comparable anti-inflammatory
activity with standard diclofenac sodium which is a chemical
analgesic at 50microL concentration (Figure 3). The Biogenic
silver nanoparticles can act as a potent anti-inflammatory Drug
(FIGURE 8).
Antimicrobial Activity:
The antimicrobial activity of the biogenic silver nanoparticles
assayed by well diffusion method against surgical site pathogen
showed minimum inhibitory concentrations at various concentrations.
The antimicrobial activity increases with increase in concentration.
The antimicrobial activity was evaluated by comparing the
zone of inhibition of the AgNPs to standard antibiotics(FIGURE
9). The antibacterial activity of AgNPs against Pseudomonas sp
was highest at 150microL with zone of inhibition measuring
17.6mm while S. Aureus and S. Mutan showed zone of inhibition
measuring 12.8mm and 11mm respectively (FIGURE 10,11,12).
Cytotoxic Activity:
The cytotoxic activity of these biogenic AgNPs were very less, indicating
that they are biocompatible. At 50 microL concentration,
there was 10% morality when the lethality assay was performed.
The antimicrobial activity of the biogenic silver nanoparticles
coated silk sutures assayed by well diffusion method against surgical
site pathogen showed zones of inhibition for different bacterias.
The zone of inhibition around the suture was 5mm when
subjected to Pseudomonas sp. While the zone of inhibition was 6mm against S. Mutans and 5mm against S. Aureus (FIGURE
11). There was no zone of inhibition identified around the Control
Group (suture with no coating). This was statistically significant
with p value -0.000.
Discussion
Foreign suture materials are a source of infection, as bacteria gets
lodged on to the suture material used when periodontal and oral
surgical procedures are performed [20]. Sutures are always placed
in areas with high vascular supply to promote healing, which is
also a moist bacteria rich environment. When natural, multi braided
sutures such as silk are used, they increase the risk of infection
as bacteria gets retained in the interstices. Thus the choice of selection
of the type of suture used proves to be a critical factor in
deciding the level of risk of infection. The use of monofilament
sutures composed of animal intestines are being restricted in the
European countries and Japan due to the allergic reaction caused
by them [22, 27]. One the other hand, the silk sutures made of
natural fibers absorb more water and favour bacterial colonization [23]. Even though both the natural sutures have good biocompatibility,
less tissue reaction and better flexibility, surgeons prefer
the use of synthetic sutures which does not favour the growth
of bacterial colonies on their surface [28]. We believe that use of
bionanocomposite coated silk sutures can reduce the colonization
of pathogenic organisms. In our current study, silver was chosen
as a non-toxic, safe inorganic antibacterial agent that has been
reported to have inhibited more than 650 types of disease causing
pathogens [24]. Moreover, silver nanoparticles have also been
reported to inhibit bacterial colonization.
Although many studies have been conducted to evaluate the effectiveness
of silver nanoparticles, their mechanism of action is
not completely understood. It is believed that the interaction of
the negatively charged bacterial cells and the positively charged
nanoparticles is responsible for the antibacterial activity of the
nanoparticles [29]. AgNPs can break the disulphide bonds of the
protein of the bacteria thereby producing an antimicrobial effect
on a wide range of gram positive and gram negative bacteria.
Studies performed by Dakal et al and Wanj et al had reported that
the AgNPs neutralizes the surface electric charge of the bacterial
membrane leading to the leakage of the cell contents and ultimately
the death of the bacteria [30, 31]. The size of the nanoparticles
are comparatively very small compared to the bacterial cell
which increases the chance of interaction with the bacteria more
frequently. The size and shape of the nanoparticles are of great
importance as they can be altered to provide desirable results [10].
The development of eco-friendly processes for synthesis of nanoparticles
is a crucial factor in the field of nanotechnology [32].
The green synthesis of nanoparticles using eco friendly agents can
make an environmentally and economically stable method which
can be cost efficient and can have additional combined efforts
of the herbs such as Solanum nigrum and Indigofera tinctoria. It
has been reported that these herbs possess antimicrobial activity,
antioxidant activity, cytotoxic properties, diuretic property, antiulcerogenic,
anti inflammatory and hepatoprotective properties.
Significantly many components of these herbs have been utilized
for bioreduction and formation of nanoparticles [15-20].
Studies performed by Sarai et al, have reported that the silver
nanoparticles produced by using the herb Chenopodium ambrosioides
as bioreducing agent has antimicrobial properties which
can be coated on sutures to reduce bacterial load on the surgical
site post operatively. Though the previous study has not mentioned
the cytotoxic properties of the nanoparticles and its herb
availability [33]. Our current study has been conducted with abundantly
available herbs, Solanum nigrum and Indigofera tinctoria
in the indian soil. Our current study has also demonstrated about
the anti inflammatory and cytotoxic properties of the same nanoparticles
synthesis by green chemistry and validates its application
in the field of oral surgery on sutures.
The increased surface area to volume ratio has favoured the Ag-
NPs to be effice in small quantities [34]. Henceforth, we contemplate
the use of biogenic nanoparticles as an alternative approach
to battle the bacterial resistance towards conventional antibiotics
[35-40]. Conventional antibiotics have to be used in minimal proportion
as exaggerated prescription of antibiotics would affect
the systemic health of the patient. Further in vivo studies have
to be performed in future to market the product in the current
scenario.
Conclusion
Aseptic field is considered as the most important pillar of oral
surgery and oral surgical procedure and it is essential to implement
all means to achieve it. The biogenic silver nanoparticles
present here can be a useful tool against the resistance bacteria
and to battle antibiotic resistance. The biogenic silver nanoparticles
synthesised with help of combined bioreducing agents such
as Solanum nigrum and Indigofera tinctoria turned out to be the
most appropriate reducing agent to coat the surgical silk sutures
with AgNPs. The formed nanoparticles inherited important antibacterial
properties against Pseudomonas sp, streptococcus mutan
and staphylococcus aureus. They also possess antiinflammatory
activity and also prove to be biocompatible. This breakthrough in
the field of surgical biomaterial may help reduce the toxic effects
of pathogenic organisms while representing an environmentally
friendly material which would not only be economically efficient
but also proves to be a material with minimal risk to mankind and
the environment.
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