(2024) 18:163
Mostafa et al. BMC Chemistry
https://doi.org/10.1186/s13065-024-01263-7

BMC Chemistry

RESEARCH

Open Access

Laser enhanced photothermal effectof silver nanoparticles synthesized by chemicaland green method on Gram‑positive
and Gram‑negative bacteria
Elham M. Mostafa1, Y. Badr1, Marwa A. Ramadan2, Mohamed M. M. Hashem3, Khaled Abo‑El‑Sooud3,
Heba N. Deif4 and Amna H. Faid1*

Abstract
Purpose The antibacterial properties of silver nanoparticles (AgNPs) are extensively identified. In large quantities,
they might be harmful. So many fields of nanotechnology have shown a great deal of interest in the developmentof an environmentally friendly, efficient method for synthesizing metal nanoparticles. Because of its antibacterial
and antifungal properties toward a wide range of microbes, chitosan silver nanoparticles (AgNPs@Cs) constitutea newly developing class of bio-nanostructured hybrid materials. Furthermore, the use of photothermal therapy (PTT)
has been suggested as a means of elimination of germs. These light-stimulated treatments are minimally invasiveand have a few side effects. In the present work, the antibacterial effect of AgNPs at low concentrations; preparedby chemical and green methods as antimicrobial and photothermal agents in photothermal therapy; with laser irra‑
diation were explored as combined treatment against MRSA, Pseudomonas aeruginosa, and Klebsiella pneumoniae.
Methods Silver nanoparticles were produced in two ways. First, by sodium borohydrides, second, by chitosan (asa natural eco-friendly reducing, and capping agent). The nanostructure of AgNPs and AgNPs@Cs was confirmed
by UV–visible spectrometer, transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIRs),
and direct light scattering (DLS). The antibacterial activity of the prepared nanoparticles and the laser irradiation
was tested against three bacterial species of zoonotic importance; MRSA, Pseudomonas aeruginosa, and Klebsiellapneumoniae; and was evaluated by measuring their minimum inhibitory concentrations (MIC).
Results Silver nanoparticles produced by the two methods had spherical shapes with nearly the same particle size.
The analysis of DLS showed that AgNPs were very stable with zeta potential − 28.8 mv, and 47.7 mv by chemical
and chitosan synthesis, respectively. Furthermore, AgNPs@Cs showed higher antibacterial activity toward the tested
bacterial species than AgNPs by chemical method. Additionally, the bacterial viability using photothermal laser
therapy was reduced compared to laser and AgNPs alone. The bactericidal activities were higher when laser diodewas coupled with AgNPs@Cs than by chemical reduction.
Conclusion The laser combined treatment had a higher antimicrobial effect than AgNPs alone or laser irradiation
alone.

*Correspondence:
Amna H. Faid
amna.hussein@cu.edu.eg
Full list of author information is available at the end of the article© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to theoriginal author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images orother third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
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regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of thislicence, visit http://creativecommons.org/licenses/by/4.0/.

Mostafa et al. BMC Chemistry(2024) 18:163

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Keywords Antimicrobial blue laser, Bacteria, Green nanotechnology, Multidrug resistance, Nanomedicine,
Photothermal agents, Photothermal therapy

Introduction
A major menace to public health is bacterial infection,and despite the effectiveness of conventional antibiotic
treatments usually, the widespread and excessive utilization of antibiotics has led to a significant worldwide
health crisis in the form of drug-resistant bacteria. Someprevalent microorganisms like Staphylococcus aureus,
Klebsiella pneumoniae, Acinetobacter baumannii [1],Pseudomonas aeruginosa, and Enterobacter species cangive rise to stubborn hospital-acquired infections, resulting in elevated levels of antibiotic resistance [2, 3]. The
inability of antibiotics to effectively combat infections
produced by multidrug-resistant pathogens (MDR), particularly those acquired in hospitals, has spurred extensive research to discover alternative antimicrobials thatoffer greater effectiveness and reduced resistance [4].
The host’s immune system is activated to get rid of theinvasive microorganisms when an infection arises. But
occasionally, severe, and uncontrollable inflammatory
reactions can occur, which can result in diseases including multiple organ failure, sepsis, and even death. Therefore, the development of novel methods to attack diverse
bacterial diseases is imperative. Drug-resistant bacteria
may now be effectively treated using antibacterial nanoparticles as an alternative. They have several exceptional
qualities, such as readily changeable surfaces and physicochemical features that may be adjusted, such as shape,
particle size, surface charge, and composition. These
attributes have great promise for treating many diseases.
Many scientists have worked on creating different antibacterial nanomaterials, including metals, silica nanoparticles, metal oxides [5], and nanoenzymes.
Nanoparticles (NPs) are novel, potential medication delivery systems that have shown promise in thetreatment of several illnesses [6]. This potency is associated with the capacity to surmount limitations suchas limited bioavailability, rapid drug excretion fromthe body, nonspecific distribution, and poor cellular permeability [7, 8]. In the developing domains ofnanomedicine, nanobiotechnology, and nanotoxicology, nanomaterials are more significant. On the other
hand, NPs have been used as therapeutic agents against
pathogenic microorganisms in the field of toxicity [5,9–11]. To comprehend the impact of nanoparticles onbacteria, fungi, parasites, and other microorganisms, itis crucial to investigate the characteristics of these particles and how they are used in various biological andmedicinal contexts. Additionally, the kind of cappingand stabilizing materials that are utilized to prepare
NPs is crucial since it influences how effective the NPs
are against microbes. They are commonly regarded asa venue for focused drug delivery, clinical diagnostics,
and medical imaging. The common inorganic nanoparticles used are olive oil, zinc oxide, silver [12], ironoxide, gold, and titanium. Green nanotechnology as an
emerging alternative technology is created on the ecological synthesis of nanoparticles by using the capability of naturally occurring materials to produce various
nanoparticles through implementing sustainable processes [7, 10, 13, 14].
When creating safe and useful nanomaterials for
medical purposes, the toxicity of silver nanoparticles onlive cells must be considered. Ag nanoparticle toxicity
to living cells depends on several variables, including
the particles’ size, shape, surface area, and concentration. Due to their higher surface area and easier penetration of cell membranes, smaller nanoparticles are
generally more hazardous than their bigger counterparts. The concentration of Ag nanoparticles can also
have an impact on their toxicity to living cells. Higher
Ag nanoparticles’ concentrations have the potential toincrease toxicity and vice versa. It’s unclear exactly how
Ag nanoparticles causes harm to living cells. Ag nanoparticles, however, can cause oxidative stress in cells,
which can harm lipids, proteins, and DNA. In the end,
this may result in cell malfunction or death [15].
For these nanoparticles to effectively limit the bacterial development, they must have a strong synergistic relationship with the biological host. There are
now three main pathways. They have been shown to
work together or individually to confer antimicrobialactivity to AgNPs. First, AgNPs act at the membranelevel, as they can cross the outer membrane and accumulate on the inner membrane, where nanoparticle
attachment to cells can cause instability and damage,thereby increasing membrane permeability and affecting leakage of cellular contents. And ultimately leads
to cell death. Additionally, AgNPs have been shownto interact with sulfur-containing proteins in bacterialcell walls, potentially disrupting the cell wall throughstructural damage. According to the second mechanism, nanoparticles can enter cells and change theirstructure and permeability by breaking through the cell
membrane. It has been suggested that AgNPs’ uniqueproperties will allow them to interact with phosphorus or sulphur groups found in intracellular material,Mostafa et al. BMC Chemistry(2024) 18:163

such as DNA and proteins, to modify their structureand functions. Similarly, by reacting with thiol groups
in the enzymes that produce reactive oxygen species
and free radicals, they can modify the respiratory chain
in the inner membrane, causing damage to intracellular machinery and initiating the apoptotic process.
The release of silver ions from the nanoparticles is athird process that is suggested to happen concurrently
with the first two, which due to their size and charge,
they can interact with cellular components and altermetabolic pathways, cell membranes and even genetic
material [5, 16]. The progress in nanotechnology has
unveiled fresh possibilities in the realm of nanomedicine, enabling the creation of nanoparticles that can be
structured into intricate designs. Lately, of blue lighthas drawn attention to the development of new phototherapeutic antimicrobial agents that exhibit significant antimicrobial activity against a variety of bacterial
and fungal pathogens but have a lower probability ofresistance compared with antibiotics. Blue light with awavelength of 400–500 nm has been shown to be very
effective against both Gram-negative and Gram-positive bacteria [17]. Photothermal therapy (PTT) is anantibacterial approach derived from thermotherapy,
utilizing various laser light sources such as visible,
near-infrared (NIR), and ultraviolet (UV) to generateheat [18–20]. PTT relies on the deployment of photothermal agents (PTAs) that generate heat when exposed
to electromagnetic radiation, leading to cell membrane
disruption, protein denaturation, and irreversible cell
damage [19, 20]. When metal nanoparticles (MNPs)
or metal oxide Nanoparticles are employed as photothermal agents (PTAs), they induce a phenomenon
referred to as “localized surface plasmonic resonance”(LSPR) [9, 21], resulting in elevating of the nanoparticle temperature. The photothermal therapy represents
a novel and efficient sterilization technique in whichmaterials transform light energy into heat energy, effectively eliminating planktonic bacteria [22]. Metal nanostructures are the focus of significant interest owing
to their distinctive characteristics [9, 21, 23]. Silver
nanoparticles (AgNPs) represent a promising biocide,reported to exhibit lower toxicity in comparison withsilver ions [13, 24–27]. It is contributed into a varietyof antimicrobial applications such as bandages, surface
coatings, medical devices, food packaging, functional
textiles, and cosmetics [28]. Silver is recognized for its
antimicrobial properties against a broad spectrum of
microorganisms and has proven a high efficacy in bactericidal applications [25, 29]. Various theories havedescribed the mechanism underlying the antibacterial
action of AgNPs, including membrane damage, cellular content leakage, the production of reactive oxygen

Page 3 of 14

species, disruption of DNA structure, and enzyme
inactivation [30]. Due to the environmental concernson the risks results from using of chemical-based synthetic methods, applying biological approaches as anappreciated alternatives have become a research hotspot for the biosynthesis of AgNPs [20]. Chitosan (Cs)
is a naturally occurring polysaccharide composed of a
combination of (1–4)-linked d-glucosamine (deacetylated unit) and N-acetyl-d-glucosamine (acetylatedunit) building blocks. Extensive research has unveiled
that Cs is a natural cationic biopolymer possessing
outstanding attributes such as exceptional biocompatibility, biodegradability, non-toxicity, bioactivity,
and the presence of multifunctional groups [9, 18, 31,32]. Owing to its diverse array of exceptional qualities,
chitosan finds extensive application in various fields,
including food packaging films, bone replacements,
artificial skin grafting, biomedical applications, pHsensitive drug delivery, and numerous other uses [18,32, 33]. Chitosan hydrogels offer several advantages
in various fields. They are commonly used in chemicalchemistry and experimental surgery, and for inhibitionof matrix proteinases. Additionally, chitosan dressingsplay a beneficial role in homeostasis and angiogenesis.
Chitosan also activates microphages, enhancing theirtumor-killing activity and promoting the constructionof interleukin-1. Furthermore, chitosan finds applications in biomedical contexts, including wound healing,
drug delivery systems, gene delivery, and tissue engineering [32, 34].
The unique physical, chemical, and biological characteristics of silver nanoparticles allow them to engage withbacterial envelopes and prevent bacterial development.
Their antibacterial and antiviral properties are attributed
to their ability to produce active radicals [15, 27]. Becauseof the uncontrolled use of antibiotics, many bacterial species developed antimicrobial resistant.
Pseudomonas aeruginosa causes diseases in humans
and a significant loss in productivity and continues topose a serious threat to the expansion of this essential
business, as well as to the environment and public health.
It also creates a range of virulence factors that impact
humans and several animal species, including fish [35].
MRSA represents a serious health hazard to both animals and humans. In human, it can cause abscesses andseptic wounds [36]. Numerous animal species, including
dogs, cats, rabbits, horses, cattle, pigs, and poultry, areaffected by MRSA. It can also infect healthy carriers [37].
By rupturing cellular membranes and increasing cellular protein leakage, silver nanoparticles (AgNPs) exhibit
remarkable antimicrobial properties against Klebsiellapneumoniae. This also reduces the production of extracellular polymeric substances (EPS), which are essential

Mostafa et al. BMC Chemistry(2024) 18:163

Page 4 of 14

for the formation of biofilms, thereby inhibiting K. pneumoniae’s ability to form biofilms. AgNPs cause multidrug-resistant K. pneumoniae to exhibit an antibacterial
action mechanism similar to that of triclosan [38].
In the present study, we conducted a comparative
investigation using both chemically synthesized andgreen-synthesized silver nanoparticles as photothermal
agents for photothermal therapy towards Gram-positive
and Gram-negative bacteria of medical importance.

Transmission electron microscope (TEM)

Materials and methods

Dynamic light scattering

Preparation of silver nanoparticles (AgNPs)

Silver nanoparticles were prepared by two different
methods. The first one is chemically by using sodium
borohydride reduction, and the second method is biosynthesis via chitosan as a reducing and a capping agent.
Synthesis of AgNPs by a modified chemical method

The synthesis of AgNPs was done according to the chemical reduction methods described by [20, 25, 27, 39], witha slight modification to get smaller particles with narrower size distribution. Briefly, a mixture of 0.08 gm oftrisodium citrate and 0.2 g PVP, has been dissolved in
10 ml water, 0.127 g silver nitrate was dissolved in 75 mlof distilled water, then added to the above mixture with
continuous steering for 15 min. An amount of 0.0378 gof sodium borohydride (the reducing agent) dissolved in
10 ml cold distilled water was added quickly to the mixture, with continuous stirring for approximately 20 min.
Then, the alteration of silver nitrate to silver nanoparticles was specified by varying the solution color to darkbrown.
Synthesis of AgNPs@Cs by chitosan reduction

In the present experiment, the synthesis of AgNPs wasdone according to the method described by [40], with aslight modification to obtain a narrower size distribution.
Briefly, 1 g of medium molecular weight chitosan was
dissolved in a 1% acetic acid (v/v) solution. Eight ml silver nitrate solution (8.89 mg/ml) was mixed with 20 mlof chitosan solution (6.92 mg/ml) and stirred for 30 min
until being homogenous. Next, the above mixture wastransferred to a beaker, and left at 95 °C for 12 h. Within,
few hours after the reaction, the solution changed fromcolorless to light yellow and finally, to yellowish browncolor, indicating the formation of AgNPs.
Characterization of AgNPs
UV–Vis spectroscopy measurements

That was recorded via Cary 5000 (UV–Vis spectrophotometer, Varian) in a wavelength ranging from 200 to
800 nm.

A high-resolution transmission electron microscope(HR-TEM, Tecnai G20, FEI, Netherlands) was used for
imaging. A drop of diluted sample solution was placedon a copper grid coated with amorphous carbon.
Bright-field imaging mode was done with an Eagle CCD
camera using a lanthanum hexaboride (LaB6) electron
source gun and an electron acceleration voltage of 200
kV.
The measurements were performed via a Zeta-Sizer
Nano-ZS (Malvern Instruments Ltd, UK) equippedwith a 633 nm laser. This analysis provides the size distribution and polydispersity index (PDI) of particles
within the sample. The hydrodynamic particle diametercan be obtained by the Stokes–Einstein equation.
Fourier transform infrared spectroscopy (FTIRs)

FTIRs were performed between 500 and 4000 ­cm−1 via
an FTIR spectrometer (4100 Jasco, Japan), by means ofa lyophilizer, AgNPs, and AgNPs@Cs were freeze-dried.
Bacterial cultures

The bacterial cultures used in this study are three bacterial species of zoonotic importance [Gram positive(MRSA), and Gram-negative bacteria (Pseudomonas
aeruginosa and Klebsiella pneumoniae)]. Two of thetested bacterial species are American Type Culture
Collection; MRSA (ATCC43300), and Pseudomonas
aeruginosa (ATCC9027); and Klebsiella pneumoniae
strain, which was isolated from chicken meat, identified, and deposited in the culture collection of Ain
Shams University as (CCASU-2022-44).
Minimum inhibitory concentration (MIC) and minimum
bacterial concentration (MBC) [41]For all biological assays, the plate count method wasused to control the minimum inhibitory concentration (MIC) of the AgNPs and AgNPs@Cs on the tested
bacteria growth. Each tested strain was grown overnight aerobically on Brain Heart Agar (MHA, Difco,France) at 37 °C. A single colony of each bacterial species was picked up and inoculated in Muller-Hintonbroth (MHB) medium. Pure bacteria cultures weregrown overnight and then adjusted to a concentrationof 1 × ­108 CFU/ml using the 0.5 McFarland standard.
Then, the bacterial strains were diluted to approximately ­106 colony-forming units per ml (CFU. ­ml−1).
In a 96-well microliter plate, AgNPs and AgNPs@Cs
solutions (100 μl of 97 and 78.4 µg/ml, respectively)
were double fold serially diluted with sterile distilledMostafa et al. BMC Chemistry(2024) 18:163

water before being pipetted into wells No. 1 throughNo. 12 of column A. The following step involved adding 100 μl of each tested bacterial culture to well No.
A1 such that the total volume was 200 μl and the concentrations for AgNPs and AgNPs@Cs solution were48.5 and 39.2/ml, respectively. Well No. A11 was used
as a negative control containing only Ag NPs, while thewell No. A12 was used as a positive control containing
only the bacterial culture. These procedures were carried out for every bacterial species. After covering theplates, they were incubated in a shaker incubator for 20
min at 37 °C then, 30 μl from each well was spread on
Mueller–Hinton agar plates (10 μl/plate) and incubatedat 37 °C for 24 h to determine the bactericidal effects.
The lowest concentration of AgNPs and AgNPs@Cs in
the sequences preventing the progress of the bacteria
in vitro was measured as the MIC.
In vitro laser treatment

For laser irradiation, a DPSS laser with a wavelength of
405 nm and 150 mW was used. Overnight cultured bacteria were collected and resuspended in fresh MHB to a
final concentration of ­108 CFU/ml. A total of 100 μl ofeach bacterial culture was transferred to a 96-well plate,
and the irradiation was done in a time series (0, 2, 4, 6,
8, 10, 12, 14, 16, 18, and 20 min). After irradiation, 30 μlfrom each well was spread on Mueller–Hinton agar
plates (10 μl/ plate) and incubated at 37 °C for 24 h to test
the inhibitory effect of the laser treatment.
In vitro photothermal treatment

To test the enhancing efficiency of laser on AgNPs and
AgNPs@Cs, the cultured bacteria were collected separately, and resuspended in fresh MHB broth to a final
concentration of 1
­ 08 CFU/ml. A total of 100 μl of thebacterial culture was treated; separately; with the lowest concentration of silver nanoparticles that was previously recorded to decrease the bacterial growth, weretransferred into a 96-well plate and each test bacteria was
incubated with AgNPs and AgNPs@Cs, independently
for 20 min irradiated with a time series (0, 2, 4, 6, and8 min). After irradiation, 10 μl from each well was spreadon Mueller–Hinton agar plates (MHA) and incubatedat 37 °C for 24 h to evaluate the combined effect of both
treatments.
Statistical analysis

The statistical analysis was carried out using R software
version R-4.3.2. The analysis was carried out to evaluate
the effect of the photo-thermal therapy, nanoparticles,
and the combined effect of both photo-thermal therapy
and the nanoparticles.

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Fig. 1 UV–visible absorption spectra of AgNPs and AgNPs@Cs

Results and discussion
UV–VIS absorption and TEM of AgNPs and AgNPs@Cs

The UV–VIS absorption spectrum exhibits the representative Surface Plasmon Resonance (SPR) band of
AgNPs, centered at approximately 410 nm as shown in
Fig. 1 signifying the formation of AgNPs, and indicating that the prepared AgNPs by the two methods had
spherical shape and slightly of the same size. In addition, the SPR band of AgNPs by chitosan was more
broadening with a slight increase in its intensity compared with AgNPs by chemical method. That may
be due to the polymeric stabilization of chitosan on
AgNPs compared to steric stabilization of AgNPs by
the chemical method. Additionally, the concentrationof the obtained AgNPs by chitosan is slightly more thanthat by chemical method. Surface Plasmon Resonance(SPR) bands were detected at 410 nm, which approves
the formation of AgNPs in chitosan. Chitosan is anextremely powerful polymeric chelating agent for silver
ions because its amino groups have a significant affinity to form a complex with metal ions. In the current
system, the complex of silver ions with amine groups inchitosan, chitosan’s reduction of the metal precursor,
and incorporation of the resultant silver nanoparticles
into a biopolymeric matrix formed by chitosan’s oxidation all led to the spontaneous generation of silverimpregnated chitosan [42].
The TEM images are demonstrated in Fig. 2a, b. These
images show that the prepared AgNPs and AgNPs@Cs
were spherical with small size distribution of approximately 10 ± 2 nm and 13 ± 2 nm, respectively with little to no aggregation between the synthesized AgNPs
and AgNPs@Cs, which implies the repulsion of the prepared nanoparticles to avoid aggregation by steric andpolymeric stabilization, respectively.

Mostafa et al. BMC Chemistry(2024) 18:163

Page 6 of 14

Fig. 2 Transmission electron microscopy images of the size distributions of a AgNPs and b AgNPs@Cs

Zeta‑potential and particle size distribution using dynamic
light scattering (DLS)

Direct measurements of light scattering and zeta potential were conducted to evaluate the hydrodynamic
size and the surface charge of the colloidal AgNPs and
AgNPs@Cs. Typically, nanoparticles with zeta potentials
exceeding + 20 mV or falling below − 20 mV demonstrateenough electrostatic repulsion among similarly charged
particles, ensuring their stability [19]. The results for the
AgNPs’ particle size and zeta potential were as follows:
the AgNPs@Cs exhibited a particle size of 123.5 nm anda zeta potential of 47.7 mV. In contrast, AgNPs exhibited a particle size of 105.9 nm and a zeta potential of− 28 mV, as shown in Fig. 3 and Table 1. It’s important
to note that the hydrodynamic size is larger than what
TEM characterization revealed, likely due to the presence of hydrated molecules surrounding the water-soluble AgNPs’ core. The zeta potential serves as a pointerof stability for nanoparticles. The negative zeta potential
value observed for the AgNPs synthesized through citrate reduction (− 28 mV) can be attributed to the existence of three deprotonated anionic carboxyl groups from
citrate ions, promoting repulsive interactions among
nanoparticles and effectively preventing AgNPs agglomeration. The table also indicates that the Polydispersity
Index (PdI) values for AgNPs produced via citrate andchitosan reductions were 0.3 and 0.4, respectively. These
values indicated the homogeneity of the resulting particles, with citrate synthesized AgNPs tending to exhibit
slightly greater homogeneity.
Chemical bond composition via FTIR

FTIR analysis detecting the various functional groups
responsible for reducing Ag+ ions and capping/stabilizing agents. As depicted in Fig. 4a, the spectra of
AgNPs@Cs solutions exhibited distinctive bands associated with the reduction and capping of silver ions bychitosan. The AgNPs@CS spectrum revealed a bandat 3442 ­cm−1, corresponding to the N–H stretching of
the amino group, while the bands observed around the2922 ­cm−1 region indicated the C–H and C–H stretching vibrations of aromatic compounds. Moreover, thebands at 1635 ­cm−1 correlated with (NH) C=O stretching vibrations. Additionally, the band at 1530 ­cm−1 werecorresponded to the binding vibrations of amide I andamide II, respectively. Furthermore, the bands at 1460
and 1057 ­cm−1 were assigned to N–H and C–N stretch
vibrations of amines, while the band at 652 ­cm−1 corresponded to the C–H bending vibrations of stabilized
silver nanoparticles [43, 44]. The presence of N–H, andC–N absorptions may be attributed to result from theinteraction between amino groups and the metallic surface of AgNPs, where the amino groups performed as
capping agents to stabilize AgNPs@Cs [43].
FTIR spectra of AgNPs were revealed in Fig. 4b. The
peaks at 1078 ­cm−1 for –C=O stretch for carboxylic
acid and 1850 ­cm−1 –C–OH stretch assigned for carboxylic acid from tri-sodium citrate as capping agent[45]. The absorption peak at 1378 ­cm−1 were corresponding to the N­ O2 compound [46], and the bands
at 3429 and 2919 ­cm−1 are assigned to O–H and C–Hstretches, respectively. The band at 832 ­cm−1 is attributed to C=C bending alkene, the C=O groups of pure
PVP showed a prominent peak at 1646 ­cm−1. These
characteristic bands can be investigated to explore theinteraction between PVP and Ag metal ions [47].

Mostafa et al. BMC Chemistry(2024) 18:163

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Fig. 3 The size distribution and zeta potential of a AgNPs@Cs and b AgNPs

Table 1 The particle size (nm), and zeta potential of AgNPs by
citrate, and chitosan reduction
AgNPs

Average particle
size (nm)

PDI

Zeta potential (mV)

AgNPs

105.9

0.308

− 28.8 mv

AgNPs@Cs

123.5

0.460

47.7 mv

The effect of AgNPs and AgNPs@Cs on the tested bacterial
strains

The inhibition effect for different concertations of
AgNPs and AgNPs@Cs (1.5, 3, 6, 12, 24 and 48.5 µg/ml)
and (0.6, 1.2, 2.4, 4.9, 9.8, 19.6, and 39 µg/ml) on MRSA,
Pseudomonas aeruginosa, and Klebsiella pneumoniae

are shown in Fig. 5. The results show a reduction in
viability reached (0, 0, 9.2, 28, 29, 22.6 and 0%), (1, 2,
10.7, 18.5, 10.2, 2.5 and 0%), and (0, 1.2, 2.4, 6, 13.3,
21.7 and 26.5%) on MRSA, Pseudomonas aeruginosa,
and Klebsiella pneumoniae, respectively. Additionally,
there was a variation in the microbial reduction with
AgNPs by chemical method as the highest concentration didn’t induce the highest inhibition, as the maximum inhibition concentration was (12,6 and 48.5 µg/ml) on MRSA, Pseudomonas aeruginosa, and Klebsiellapneumoniae, while, AgNPs prepared by chitosan results
show a reduction in viability which reached (35, 47, 78,
100, 100, 100 and 100%), (33.6, 49.5, 94, 100, 100, 100
and 100%), and (11, 31.3, 49.4, 100, 100, 100 and 100%)

Mostafa et al. BMC Chemistry(2024) 18:163

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Fig. 4 FTIRs of a AgNPs@Cs, b AgNPs

Fig. 5 Viability growth inhibition at different concentrations of Ag NPs by citrate and AgNPs@Cs on a Pseudomonas aeruginosa b Klebsiellapneumoniae and c MRSA

Mostafa et al. BMC ChemistryFig. 5 continued(2024) 18:163

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Mostafa et al. BMC Chemistry(2024) 18:163

on MRSA, Pseudomonas aeruginosa, and Klebsiellapneumoniae, respectively.
Numerous investigations have demonstrated the antibacterial action of AgNPs. Others have demonstrated themechanism of their contact with bacterial cells. AgNPs;
according to one theory; are just sources of silver ions,which they deposit onto the cell wall where they attach tothe cytoplasmic membrane [48]. Silver ions are thought
to react with the thiol groups of protein molecules, disrupting enzyme function and sometimes resulting incell death. Figure 5 illustrates how AgNPs themselves
have a greater antibacterial action. Because AgNPs arebigger than silver ions. For that, AgNPs are prepared toget more surface area, consequently, interact with more
molecules and thus exhibit greater antimicrobial activity [49]. As shown in Fig. 5, in all tested concretions of
AgNPs@Cs, there was a significant reduction in viabilityon the three tested microbes which may be due to thatthrough a variety of methods. Chitosan can prevent thegrowth of bacteria. The most basic one is an electrostatic
contact between the positively charged N­ H3+ sites of chitosan and the negatively charged membranes of microorganisms. Intracellular material is released because of
the contact changing the microbial cell permeability. It
has also been shown that chitosan is an efficient way toFig. 6 Laser irradiation effect on the tested bacterial species

Page 10 of 14change the calcium content of cell walls. In this instance,
chitosan alters the osmotic equilibrium of the membrane
wall and interacts with the stability of peptidoglycan toprovide its antibacterial action. Chitosan can also affectthe electron transport chain and oxygen reduction mechanisms, which can compromise the membrane’s energy
stability. Another proposed mechanism is related to theability of chitosan to cheat metal ions, as it disrupts theintegrity of microbial membranes. Positively charged chitosan can also block RNA and protein synthesis, thereby
inhibiting bacterial growth. The antibacterial characteristics of AgNPs decreased with chitosan may provide an
explanation for this phenomenon. First, it is thought thatelectrostatic contact accounts for most of the interaction between bacterial cells and chitosan. The protonatedamino group of chitosan’s positive charge interacts with
the negatively charged molecules on the bacterial cellsurface, causing the cell surface to be permeable, which inturn allows internal chemicals to seep out and ultimately
results in the death of the bacterial cell. Furthermore,
it has been reported that the antibacterial mechanismof AgNPs@Cs results in alterations to the membranepotential and permeability. These modifications upset the
homeostasis of the cell and ultimately cause bacterial cell
death. This could account for the greater inhibition effectMostafa et al. BMC Chemistry(2024) 18:163

of AgNPs@Cs when compared to AgNPs through chemical reduction [50].
Effect of laser irradiation on the tested bacteria

Calculating the CFU of the tested bacteria before andafter laser treatment allows one to assess the inhibitory
effect of laser light (405 nm) on MRSA, Pseudomonas
aeruginosa, and Klebsiella pneumoniae for 2, 4, 6, 8, 10,
12, 14, 16, 18 and 20 min, as represented in Fig. 6. MBC
were reached at 12, 16 and 16 min which are equivalent
to the exposure doses 262.8, 350 and 350 J/cm2, while
MIC was 10, 14, and 14 min which is equivalent to the
exposure doses 219, 306.6 and 306.6 J/cm2 for MRSA,
Pseudomonas aeruginosa, and Klebsiella pneumoniae,
respectively.
Assessment of photothermal effect on the tested bacteria

The growth pattern of MRSA, Pseudomonas aeruginosa,
and Klebsiella pneumoniae in the presence of AgNPs and
AgNPs@Cs with 405 nm (150 mW) laser irradiation are
exposed in Fig. 7a, b, respectively. It was carried out totest the photothermal effect against the three tested bacterial species. In this step, we used the safe lowest concentration (1.5 and 1.225 µg/ml) for AgNPs and AgNPs@
Cs, respectively and irradiated for 0, 2, 4, 6, 8 min with
405 nm laser light. As shown in Fig. 7, there was a significant reduction in viability with increasing irradiation
time, in case of AgNPs by chemical method irradiatedby laser for 8 min resulted in a reduction reached (78.5,
69 and 68%) on MRSA, Pseudomonas aeruginosa, andKlebsiella pneumoniae, respectively. While, in AgNPs@
Cs irradiated with the same laser light reached MBC at 6,
8 and 8 min and MIC at 4, 6 and 6 min which is equivalent to the growth inhibition of 72, 88 and 79% on MRSA,

Fig. 7 Photothermal effect by a AgNPs & b AgNPs@Cs

Page 11 of 14

Pseudomonas aeruginosa, and Klebsiella pneumoniae,
respectively.
Whatever the particle size or laser energy, combining
the use of laser and AgNPs further inhibited the development of bacteria, suggesting that the combined treatmentof laser and AgNPs is superior to either therapy alone.
These bacterial inactivation methods may help to explain
our findings that 405 nm laser irradiation is antimicrobial
and that laser therapy with AgNPs treatment has evenmore potent antimicrobial activity than laser treatment
alone [18, 51]. ROS are known to cause metabolic pathway disruption, which leads to cell malfunction. AgNPs
also can adhere to cell membranes and alter the chemicaland physical makeup of cell walls, which can hinder andinterfere with essential cellular processes like respiration,
ion transport, cell division, and membrane permeability[52].
After testing the effect of the laser light on the bacterial growth, and viability, it was clear that there was a statistically significant effect of the photo-thermal therapy
by time on bacterial CFU levels across the three tested
bacterial species (the p-value for the “Timepoint” factor
is 0.00304, which represents the significance of the timeeffect. These results agreed with that mentioned by [53]who suggested that laser treatment can increase the antimicrobial efficacy of MRSA. The effective laser exposure
time for MRSA was 12 min but the effective one reportedby [54] was 5–6 min. This is also compatible with [55]who confirmed that the laser therapy was very effective
in killing of Pseudomonas aeruginosa.
In the present study, photothermal therapy is effective in killing MRSA, Klebsiella pneumoniae, and Pseudomonas aeruginosa. The effective exposure times to thelaser therapy for MRSA, Pseudomonas aeruginosa, andMostafa et al. BMC Chemistry(2024) 18:163

Klebsiella pneumoniae, were 12, 16, and 16 min, respectively. There were distinct differences among these threebacterial species, as when AgNPs were tested, it couldn’t
inhibit the bacterial growth of any of the tested bacterial species. Its starting concentration was 48.575 µg/ml,
which then was serially diluted to 1.5 µg/ml. So, AgNPs
alone were not effective, but [56] stated that AgNPs
effectively inhibited the growth of MRSA, and Klebsiellapneumoniae, which may be attributed to the difference inthe used concentrations. Conversely, the ANOVA outcomes presented the statistical significance of the difference in mean CFU counts between different groups(for each bacterial species, the AgNPs@Cs, dilution, andAgNPs@Cs, dilution). The p-value for each term indicated whether there was a significant change in mean
CFU counts between different groups. For MRSA, thep-value was significant for AgNPs@Cs (p = 0.000435);
meaning that AgNPs@Cs had a significant effect on
the bacterial growth; but not for Dilution (p = 0.157),
or AgNPs@Cs: dilution (p = 0.115). This suggested that
there was a significant difference in mean CFU counts
among different antimicrobial agents for MRSA. But,
for Pseudomonas aeruginosa, the p-value was significant
for AgNPs@Cs (p = 0.000192), and AgNPs@Cs: dilution(p = 0.0585), but not for dilution alone (p = 0.365). All theprevious results suggested that there was a significant
difference in mean CFU counts among the different antimicrobial agents, and their interaction with dilution for
Pseudomonas aeruginosa.
The AgNPs@Cs were used with a starting concentration of 39 µg/ml, which was then serially diluted to concentrations 1.225 µg/ml. All the concentrations; except
1.225 µg/ml; were effective in killing the tested bacterial
species, so, we started to test the effect of the photothermal therapy on the least ineffective concentrations;1.225 µg/ml for AgNPs@Cs, and 1.5 µg/ml for AgNPs.
The used antimicrobial agents and the time-point also
had a significant interaction effect (p-value = 0.0013561),
suggesting that the effect of the antimicrobial agent varied with the exposure time to the photo-thermal therapy.
The interaction between bacterial species, the antimicrobial agent, and the time point wasn’t statistically significant (p-value = 0.0856), indicating that the combined
effect of all these three factors wasn’t significant at theconventional significance level of 0.05. Further comparative studies between various Gram-negative and Grampositive bacteria are required to confirm this hypothesis.

Conclusion
The small extremely stable AgNPs could be prepared via
the two methods chemically, and eco-friendly methods
by chitosan as reducing and capping agent which showhigh stability with zeta potential − 28 mV and 47 mV,

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respectively. The present study is the first to prepare silver
nanoparticles by using chitosan as a reducing and a capping agent in combination with low power laser on bacteria. AgNPs exhibited a photothermal therapeutic effecton the Gram positive (MRSA), along with Gram negative
bacteria (Klebsiella pneumoniae, and Pseudomonas aeruginosa) by combining AgNPs with low power diod laser light(405 nm). That might be suitable to treat the fast-growing
drug-resistance bacteria with the slow progress of innovative antimicrobial agents. Furthermore, the assessmentof laser low-power radiation coupled with a low dose of
AgNPs has a promising effect on the tested bacterial species. The obtained data revealed that the dominant effectwas coming from AgNPs by chitosan which were effective
and stable in combination with laser, against all the tested
microbes. Future work in progress to use the prepared nanoparticles for invivo study and as antibiotic drug carriers
against more bacterial species.
Acknowledgements
None.
Author contributions
Y. Badr and M. M. Hashem considered the idea, Elham M. Mostafa preparedsamples, conducted UV–VIS, and FTIR measurements, and analyzed data withassistance from K. Abo-El-Sooud, Heba N. Deif, Amna H. Faid, and Marwa
A Ramadan analyzed the experimental data. Elham M. Mostafa wrote themanuscript and designed the Figures, with technical input from Amna H. Faid.
Collectively, the authors assisted in the data analysis and technical discussion.
Funding
Open access funding provided by The Science, Technology & Innovation
Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank
(EKB). This research neither receives any specific grant nor fund from any fund‑ing agencies in the public, commercial, or not-for-profit sectors.
Availability of data and materials
The datasets used and/or analyzed during the present study were obtainablefrom the corresponding author on reasonable request.

Declarations
Ethics approval and consent to participate
Not applicable.
Consent for publication
The present study is available from the corresponding author upon reason‑
able request.
Competing interests
The authors declare no competing interests.
Author details
1
Department of Laser Sciences and Interactions, National Institute of Laser
Enhanced Science (NILES), Cairo University, Giza, Egypt. 2 Department of Laser
Application in Metrology, Photochemistry, and Agriculture, National Institutefor Laser Enhanced Science (NILES), Cairo University, Giza, Egypt. 3 Departmentof Pharmacology, Faculty of Veterinary Medicine, Cairo University, Giza, Egypt.
4
Microbiology Department, Faculty of Veterinary Medicine, Cairo University,
Giza, Egypt.
Received: 28 October 2023 Accepted: 5 August 2024

Mostafa et al. BMC Chemistry(2024) 18:163

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