Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 12 April 2021 doi:10.20944/preprints202104.0311.

v1

Chitosan/Silver Nanoparticle/Graphene Oxide Nanocomposites

with Multi-drug Release, Antimicrobial, and Photothermal
Conversion Functions
Zheng Sua,b, Daye Sunb, Li Zhangc, Miaomiao Hec, Yulin Jiangc, Bronagh Millar b, Paula Douglas b, Davide Mariot-
tid, Paul Maguired, Dan Sun*,b

a. Department of orthopedics, The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine,
University of Science and Technology of China, Hefei, Anhui 230001, China
b. School of Mechanical & Aerospace Engineering, Queens University Belfast, Belfast, BT9 5AH, UK
c. Research Center for Nano-Biomaterials, Analytical & Testing Center, Sichuan University, Chengdu 610065,
China
d. Nanotechnology and Integrated Bioengineering Center (NIBEC), Ulster University, Co Antrim BT37 OQB,
UK
Correspondence: [email protected]; Tel.: +44 (0) 28 90974701
Abstract: In this work, we designed and fabricated a multifunctional nanocomposite system which consists of
chitosan, raspberry-like silver nanoparticles and graphene oxide. Room temperature atmospheric pressure
microplasma (RT-APM) process provides a rapid, facile, and environment-friendly method for introducing
silver nanoparticles into the composite system. By loading different drugs onto the polymer matrix and/or
graphene oxide, our composite can achieve a pH controlled dual drug release with release profile specific to
the drugs used. In addition to its strong antibacterial ability against E. coli and S. aureus, our composite also
demonstrates excellent photothermal conversion effect under irradiation of near infrared lasers. These
unique functionalities point to it’s the potential of nanocomposite system in multiple applications areas
such as multimodal therapeutics in healthcare, water treatment, and anti-microbial, etc.

Keywords: Chitosan; Silver nanoparticles; Graphene oxide; Nanocomposites; Antibacterial prop-

erty; Drug delivery

1. Introduction
Chitosan (CS) exhibits biocompatibility and biodegradability and has been explored
widely for biomedical applications such as bone/skin regeneration, wound dressing and
cancer therapy [1-3]. Graphene oxide (GO), on the other hand, has excellent mechanical
properties and electrical conductivity [4]. Its high specific surface area and oxygen-con-
taining surface functional groups has enabled its application as drug carrier [5, 6]. In ad-
dition, GO has also emerged as promising photothermal agents for photothermal therapy
especially due to their high absorption cross-sections in the near infrared (NIR) region [7,
8]. These unique physio-chemical properties have enabled its wide applications in the bi-
omedical field, including drug delivery, photothermal therapy and tissue regeneration [9,
10].
Combining CS with GO could lead to nanocomposites with unique functionality for
wider range of applications, particularly in the biomedical field. For instance, Barra et
al.[11], developed electrically conductive chitosan - reduced graphene oxide flexible bio-
nanocomposites for food packaging and biological applications. Hermenean et al.[12], fab-
ricated chitosan-graphene oxide 3D scaffold via pi-pi interaction, where the resulting
composites has been proposed for bone regeneration. Rana et al.[13], used CS to surface
functionalize GO through chemical crosslinking, the in-vitro study showed the higher cell
viability of the functionalized GO. Bao et al.[14], fabricated biocompatible CS-functional-
ized GO, which can be used as nano-carrier for drug and gene delivery. Shamekhi et
al.[15], prepared GO containing CS scaffolds for cartilage tissue engineering with en-
hanced physical and mechanical properties via cross-linking and incorporation of nano-
particles. Liu et al.[16], obtained the three-dimensional (3D) CS/GO scaffolds with aligned

© 2021 by the author(s). Distributed under a Creative Commons CC BY license.

Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 12 April 2021 doi:10.20944/preprints202104.0311.v1

pores for tissue engineering with enhanced mechanical strength, shape-memory, cell
alignment guiding capabilities, and protein adsorption ability. The GO was also used to
improve the electrical conductivity of biomaterials in cardiac tissue engineering [17, 18].
In recent years, there is an increasing demand for multi-functional materials, partic-
ularly in the biomedical field. For instance, enabling simultaneous anti-microbial and
drug release functions is of particular interest for conditions such as osteogenesis, inflam-
mation, and infections [19, 20]. Effective anti-bacterial and drug delivery functions com-
bined with photothermal therapy may enable multimodal therapeutic functions with en-
hanced treatment efficacy towards challenging health conditions such as cancer [21, 22]
and severe arthritis [23].
Room temperature atmospheric pressure microplasma (RT-APM) process is a rapid,
facile, and environment-friendly method that can be used to prepare nanocomposites. The
resulting nanocomposites consisting of CS with embedded silver nanoparticles through
RT-APM have demonstrated tailored structure/properties as well as promising antibacte-
rial activity against E. coli and S. aureus [24]. One interesting aspect worth further inves-
tigation is the addition of other functional nanomaterials (e.g., photothermal conversion
agents) into this system to form a hybrid multi-functional nanocomposite and to further
demonstrate the potential of such hybrid nanocomposites for a wider range of biomedical
applications.
In this work, GO was introduced into the RT-APM synthesized CS/raspberry-like
silver nanoparticles (RB-AgNPs) system to produce a CS/RB-AgNPs/GO hybrid nano-
composite. The resulting hybrid composites were then characterized for their structure
and properties via SEM, TEM, UV-vis, FT-IR, and TGA, DSC, and their functionality to-
wards anti-microbial, drug release and photothermal conversion has also been demon-
strated.

2. Materials and Methods

2.1 Materials
Chitosan (CS, low molecular weight 50,000-190,000 Da, 75-85% deacetylated), silver
nitrate (AgNO3, ACS reagent, ≥99.0%), fluorescein sodium, methylene blue, and phos-
phate buffered saline (PBS) tablets (pH=7.4) were purchased from Sigma-Aldrich (United
Kingdom). Graphene oxide (5 mg/mL aqueous solution, flake size: 0.5-5 µm, 1 atomic
layer at least 60%) was purchased from Graphene Laboratories, Inc. Acetic acid (puriss,
glacial, ≥99.9%) was obtained from Riedel-de Haёn Sigma-Aldrich Laborchemikalien
GmbH. Ultrapure water (>18.2 MΩ cm Millipore Milli-Q system) was used in the all ex-
periments. Helium gas was purchased from BOC, UK. Methylene blue (MB) and fluores-
cein (FL) were obtained from Sigma-Aldrich, UK.
2.2 Fabrication CS/RB-AgNPs/GO nanocomposite
Room temperature atmospheric pressure microplasma (RT-APM) process as a rapid,
facile, and environment-friendly method was used to synthesize CS/RB-AgNPs. More
specifically, as purchased CS powder was dissolved in 2% (v/v) acetic acid and stirred for
24 hours under room temperature to obtain a 2% (w/v) homogeneous solution. Appropri-
ate amount of AgNO3 (50mN) was added to the CS solution to obtain precursor solution
CS+AgNO3 mixture. The mixture was subject to RT-APM treatment see in Scheme 1 (a),
gives the details of RT-APM setup. A stainless steel capillary cathode (0.25 mm inner di-
ameter and 0.5 mm outer diameter) was placed ~ 1.5 mm above the solution surface, and
a carbon rod (anode) was immersed in the liquid. Helium gas was passed through the
cathode capillary at a flow rate of 25 SCCM, a constant current of 5 mA and voltage of 0.7-
1.0 kV was maintained during the plasma treatment. The RT-APM treatment was carried
out for 10 min and the samples were stirred during the treatment to obtain a well-mixed
colloid (see Scheme 1 (b)). The resulting sample is named as CS/RB-AgNPs.
The CS/RB-AgNPs/GO nanocomposite with 0, 0.5, and 1.5 wt% GO were prepared
by adding appropriate amount of GO into the RT-APM treated CS/RB-AgNPs sample. All
liquid samples were then transferred into a Teflon mould (with 9 mm diameter × 4 mm
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 12 April 2021 doi:10.20944/preprints202104.0311.v1

recess) and the samples were frozen over night at -35 °C, followed by freeze-drying
(Lyotrap LTE) for 24 hours at -60 °C. Table 1 shows the composition and nomenclatures
of the samples prepared in this study.
2.3 Characterization
Fourier transform infrared spectrometer (FTIR, Perkin Elmer Spectrum 100 ATR, 4
cm-1 resolution and 36 scans per sample) was used to analyse the samples. Thermograv-
imetric analysis (TGA, TA Instruments SDT-Q600) was carried out under nitrogen atmos-
phere over the temperature range 40 - 600 °C at 10 °C per min increment. The morphology
of the cryo-fractured sample (gold sputtered) was observed by scanning electron micros-
copy (SEM, HITACHI FlexSEM 1000) with acceleration voltage in the range of 5-15 kV.
Differential Scanning Calorimeter (DSC) was carried out using Perkin-Elmer DSC 6 in-
strument. The samples were first scanned from 20~110°C at 10°C/min to remove water
and eliminate thermal stress, The samples were then held at 110°C for 10 min and cooled
from 110°C to 20°C at 10°C /min. The second scan was carried out from 20°C to 380°C at
10°C/min. UV-vis spectroscopy (Cary Win UV) was used to characterize drug release un-
der pH 7.4 and pH 4, respectively. X-Ray Diffraction (XRD, PANalytical X’PERT ProMPD)
was used to analyse the crystallinity of samples. Lloyd’s LRX Tensile Tester was used for
the Quasi-static mechanical testing in compression with 50 N load cell, following ASTM
D1621-101 with specimen sizes (10 mm diameter, 5 mm height).
2.4 Drug releasing tests
Two model drugs FL and MB have been selected. To investigate the single drug re-
lease profile, FL loaded GO (1.5 mg FL per 100 mg GO), namely (GO-1.5)-FL or MB loaded
CS/RB-AgNPs (0.2 mg MB per mL of CS/RB-AgNP colloid)), namely (CS/RB-AgNPs)-MB,
was used to prepare the single drug laden nanocomposite, respectively. For dual drug
release, both FL loaded GO and MB loaded CS/RB-AgNPs were used. Three types of drug
loaded nanocomposites were prepared, namely, (CS/RB-AgNPs)-MB/GO-1.5, CS/RB-
AgNPs/(GO-1.5)-FL, and (CS/RB-AgNPs)-MB/(GO-1.5)-FL. Drug release test was con-
ducted in 10 mL PBS (pH 7.4 or 4.0, 37 °C). At different time intervals, 2.5 mL of test solu-
tion was analysed using UV-vis spectrophotometer and the remaining solution was
topped up with fresh PBS. The cumulative release percentage of FL and MB was calcu-
lated following Equation (1):
Cumulative drug release (%)=M_t/M_0 ×100% (1)
Where Mt is the amount of drug released at time t and M0 is the initial amount of
drug load.
2.5 Antibacterial tests
The antibacterial properties of the nanocomposites were evaluated by zone of inhibi-
tion test using Staphylococcus aureus (Gram-positive bacteria) and E.coli (Gram-negative
bacteria) respectively. 100 µl of the overnight bacterial culture (1 × 108 CFU/ml) was
spread on Nutrient Agar plates. The sample was then placed in the plates in triplicates
followed by incubation for 24 h at 37 °C. After the incubation period the zone of inhibition
was measured.
2.6 Photothermal conversion behaviour tests
IR thermal camera (FORTIC 220RD PCBA) is used to capture the thermal image of
samples under near infrared (NIR) 808 nm laser irradiation with different powers (0.2, 0.4,
0.6, and 1.0 W) with a light window of 50.24 mm2.

3. Results

3.1 Materials characterization

Fig. 1 (a) shows the UV-vis spectra of all testing samples. In theory, GO should have
an absorbance peak at around 200 nm due to the pi -pi* conjugation of C=C bonds [25].
However, this peak overlaps with the characteristic peak of pure CS at 200-250 nm and
cannot be easily distinguished in the spectra of the CS/RB-AgNPs/GO composite samples.
Optical images in Fig.1 (a) inset shows the change of solution colours for different sam-
ples, where brown color is indicative of presence of RB-AgNPs. The presence of RB-AgNP
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 12 April 2021 doi:10.20944/preprints202104.0311.v1

is further supported by the typical broad surface plasmon resonance (SPR) absorption (~
410 nm) for RB-AgNPs. The TEM images in Fig. 1 (b) shows the morphology and size
distribution of RB-AgNPs in CS/RB-AgNPs/GO-1.5 solution with average size of 73.69
nm.
Fig. 2 (a) presents the photographs of nanocomposites with different RB-AgNP
and/or GO contents. The TGA results provide better understanding of the thermal decom-
position and thermal stability of pure chitosan and composites. Pure CS has an initial de-
composition at 270.16 °C due to the thermal degradation of polymer chains. As shown in
Fig. 2 (b), all TGA curves have similar decomposition trend. And the weight loss around
127.32 °C is attributed to the loss of water from the polymer. Interestingly, the loading of
RB-AgNPs and GO delay the initial decomposition of the CS’s polymer chains, however,
has higher residual mass from 40.25 % to 41.83 % at 700 °C than pure CS. The decomposi-
tion hindering effect can explain this phenomenon [26].
Fig. 2 (c) shows the DSC curve of pure CS, CS/RB-AgNPs, and CS/RB-AgNPs/GO
composites obtained from the second heating. The CS chain segmental mobility could be
reflected by the glass transition temperature (Tg). The Tg of pure CS is 134.70 °C and the
broad baseline step is due to the rigidity of the CS molecular structure [27]. The addition
of RB-AgNPs increased the Tg of CS/RB-AgNPs composites to 138.89 °C, whereas the
presence of both GO and RB-AgNPs increased the Tg of CS/RB-AgNPs/GO composites to
141.91 °C. The increase in Tg is ascribed to the interaction between CS chains and GO
sheets and RB-AgNPs [28, 29]. Such interaction may constrain the segmental motion of CS
chains by hydrogen bonding and electrostatic interaction and hence increase its thermal
stability [30]. The exothermic peak around 270-350 °C shown in Fig. 2 (c) and Table S1
both indicate the thermal decomposition of composites, which can be further related to
the material weight loss seen in the TGA curves (Fig. 2 (b)).
Fig. 2 (d) presents the FT-IR spectra of all samples. The characteristic bands of amide
I and amide II groups are located at 1654 cm-1 and 1385 cm-1. The peaks at 1333 cm-1 and
1258 cm-1 belong to the O-H bending vibration, and the anti-symmetrical stretching of C-
O-C bridge can be found at 1151 cm-1 [31, 32]. The strong broad band at 3100-3650 cm-1
represents characteristic peak of N-H or O-H stretching vibration from CS, RB-AgNPs or
GO [33]. The intensity of N-H bending vibration peak at 1532 cm-1 decreases due to the
interaction between RB-AgNP and CS through hydrogen bonding [31].

3.2 Mechanical properties

Further SEM analysis in Fig. 3 shows that the influence of nanofillers on the compo-
sites’ microstructure and mechanical property (compressive modulus). Nano-sized RB-
AgNPs led to reduced pore size but greater pore density when compared with pure CS.
Whereas CS/RB-AgNPs containing GO sheets with flake size of 0.5-5 µm shows more sim-
ilar structure to pure CS. Fig. 3 (d) shows the mean compressive elastic modulus of com-
posite samples. The increased modulus for CS/RB-AgNPs may can be attributed to hydro-
gen bond formation between nanofillers and polymeric matrix, resulting in restricted mo-
tion of backbone chain [34]. When GO is introduced into the system, RB-AgNPs (or CS
encapsulated RB-AgNPs) may interact preferably with the GO flakes through physical
adsorption or electrostatic interaction [35]. This may lead to agglomeration of the nano-
fillers (also see Fig S2), which can deteriorate the composite mechanical property (reduced
stiffness).

3.3 In-vitro drug release profiles

FL and MB were selected as type model drugs as their molecular weights are similar
to some of the anti-cancer, anti-inflammatory, and bone regeneration drugs (such as Cis-
platin, Not-steroidal anti-inflammatory indomethacin, Dexamethasone, FTY20, Vitamin
D3, Sphingosine-1, etc.) [36]. The successful loading of FL onto GO (GO-FL) can be con-
firmed by the UV-vis spectroscopy. Fig. 4 (a) shows the UV-vis spectra of GO, GO-FL, and
the as-prepared FL solutions. The original characteristic peaks of FL (238, 459 nm and 491
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 12 April 2021 doi:10.20944/preprints202104.0311.v1

nm) were blue shifted to231 nm, 448 nm and 483 nm in the GO-FL sample. The character-
istic peaks of MB at 292 nm and 663 nm remain unchanged after loaded onto CS/RB-
AgNPs. These characteristic peaks were used to investigate the release of MB in solution
(Fig. 4 (b)).
For in-vitro drug release tests, we loaded MB onto CS/RB-AgNPs matrix and loaded
FL onto GO to form the two separate single drug release models. Subsequently, we com-
bined MB laden CS/RB-AgNPs and FL laden GO to obtain the dual drug release system.
Fig. 5 presents the percentage of cumulative drug released in neutral (pH=7.4) and acid
(pH=4.0) environment over 240 h. For single drug release of MB (MB on CS/RB-AgNPs)
(Fig. 5 (a)), a burst release was evident under pH 7.4 in first 10 h, followed by a more
stabilised release of 70-80% after 40-50 h. Under pH 4, however, the release rate was sig-
nificantly accelerated the drug release reached 100 % from 80 h. In contrast, the single
drug FL (FL on GO) (Fig. 5 (b)) release shows a time lag behaviour. Only 30% FL was
released in the first 10 h under pH 7.4 and the cumulative release only increased slowly
up to 45% towards the end of 240 h. The lower of pH also significantly altered the release
profile, which featured a continue increase of cumulative drug release at a much higher
rate throughout the measurement period. Fig. 5 (c) and (d) shows the drug release profile
of CS/RB-AgNPs/GO-1.5 when it acted a dual release system (MB in CS/RB-AgNPs and
FL on GO). It can be seen from Fig. 5 (c) that the addition of FL (FL-GO to replace GO)
slowed down the MB release rate for both pH conditions as compared with the single
drug (MB) release system. In contrast, when MB-loaded CS/RB-AgNPs was used, the FL
release rate from CS/RB-AgNPs/GO-1.5 was only increased slightly under both pH con-
ditions.

3.4 In-vitro drug release kinetic model

In order to obtain a suitable drug release model that best describes the drug release
profile, several kinetic models have been considered for the drug release systems involved
in this study, these include zero order (cumulative percent drug released versus time),
first order (log cumulative percent drug retained versus time), Higuchi (cumulative per-
cent drug released versus√time), and Korsmeyer-Peppas (log cumulative percent drug re-
leased versus log time) and the curve fitting results were shown in Table 2, Fig. S3 and
Fig. S4 [37]. The modelling accuracy was evaluated by comparing the coefficient of deter-
mination (R2) values. In the Korsmeyer-Peppas (Fickian diffusion) model, the mechanism
of drug release was indicated by the value of the release exponent N [38, 39]. Porous struc-
tures typically exhibit N<0.45 and diffusion occurs through water-filled pores and
through polymer layer due to the swollen matrix [40, 41].
The drug release kinetic analysis results from Table S2 suggests that the Korsmeyer-
Peppas model best describes the predominant release mechanism for both single and dual
drug systems (Fig. 6). The porous structure of the composites were corroborated by the
value of release exponent N < 0.45. When fitting the drug release curves based on the
Korsemeyer-Peppas model [42], the cumulative amount of drug release (Mt) could be de-
scribed by equation (2):
M_t=ktN (2)
Where N was the release exponent in Korsmeyer-Peppas model, indicative the drug
release mechanism, t was the time elapsed and k was defined by equation (3):
k=A√(2C_init DC_s)) (3)
Where A was the cross sectional area of the cylinder (diameter=1.0 cm, height=0.5
cm), C_init was original concentration of the drug (MB=0.2 mg/mL and FL=1.5 wt%), C_s
was the solubility (70 mg/mL for MB and 500 mg/mL for FL) and D was the diffusion
coefficient of the drug (0.42×10-5 cm2/s for pristine FL and 0.79×10-5 cm2/s for pristine
MB) [43].
For the dual drug system, the true diffusion coefficient of MB and FL is different from
the ideal diffusion coefficient due to the potential host-guest and guest-guest interac-
tions[44] between GO, MB, and FL see Fig. 7 and Table 2.
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 12 April 2021 doi:10.20944/preprints202104.0311.v1

In our drug laden composites, FL was loaded onto GO via weak pi-pi interaction,
which can be described as a weak host (GO)-guest (FL) interaction. When MB was intro-
duced into the system, the stronger ionic interaction between MB and GO (host (GO)-
guest (MB)) drove, replacement of the loosely adsorbed FL with MB, to form a more stable
structure, leading to a slower MB but greater FL release. In addition, the repulsive force
between the negatively charged functional groups on FL and GO accelerate the desorption
process of FL from GO sheet surface. The true diffusion coefficients (DK) of each system
following Korsemeyer-Peppas model were obtained through further optimization to-
wards the objective function, see Table 2.

3.5 Antibacterial ability

Inhibition zones of the composite samples were measured to evaluate their antibac-
terial ability against E. coli and S. aureus, see Fig. 8. It can be seen that pure CS exhibits
no visible antibacterial ability. In contrast, samples containing RB-AgNPs showed signif-
icantly enhanced antibacterial performance. The presence of RB-AgNPs can generate re-
active oxygen species (ROS) when in contact with the bacterial strains, and these ROS
could destabilize the bacteria plasma membrane potential, deplete the levels of intracel-
lular adenosine triphosphate, and eventually lead to the death of bacterial cells [45]. In-
creased GO content also leads to further enhanced antibacterial ability, because GO can
cause oxidative pressure [46] or bacterial cell membrane damage [47].

3.6 Antibacterial ability

All samples were immersed in PBS solution and their photothermal conversion be-
haviour under 808 nm laser irradiation have been examined, using PBS solution as the
control. Fig 9 (a) and (b) show that under 1W laser irradiation, samples containing no GO
exhibited no photothermal conversion effect. For CS/RB-AgNPs/GO composites, the sam-
ple contains greater GO content demonstrated greater photothermal conversion effi-
ciency. Fig. 9 (c) shows that by manipulating the laser power, the temperature of profile
of the selected sample (CS/RB-AgNPs/GO-1.5) can be fine-tuned. When 2 W power was
used, the sample were heated to 45 °C within 100 s, which is within the therapeutic win-
dow for photothermal therapy applications. Fig. 9 (d) further illustrates the excellent pho-
tothermal conversion stability of the composite samples during cyclic heating/cooling
tests.

4. Conclusions
In this study, we fabricated CS/RB-AgNPs/GO hybrid nanocomposite by incorporat-
ing GO into RT-APM synthesized CS/RB-AgNPs. The resulting nanocomposites have
then been characterized towards several functional applications including multi-drug re-
lease, anti-microbial and photothermal conversion. The drug loading/release behaviour
and the underlying mechanisms have been studied in detail using both single drug and
dual drug models. Our composite system demonstrated pH sensitive release and the drug
release profile can be best described by the Korsmeyer-Peppas model. The nanocompo-
site also demonstrated significant anti-microbial properties against E.coli an S.aureus bac-
teria strains as well as excellent photothermal conversion efficiency, which can be manip-
ulated by tuning the laser power. These promising functionality may lead to the potential
application of our nanocomposites in multiple fields, such as multi-therapeutic platform
for the treatment of certain cancers, anti-microbial membranes/coatings, water treatment,
etc.

Acknowledgments: The authors would like to acknowledge the financial support from China Schol-
arship Council (201806340039, 201606170059), China Postdoctoral Science Foundation
(2020M671893), the Fundamental Research Funds for the Central Universities (WK9110000155), and
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 12 April 2021 doi:10.20944/preprints202104.0311.v1

the Engineering and Physical Sciences Research Council (EPSRC) (EP/P00394X/1, EP/M015211/1 and
EP/R008841/1).
Conflicts of Interest: Declare conflicts of interest or state “The authors declare no conflict of inter-
est.” Authors must identify and declare any personal circumstances or interest that may be per-
ceived as inappropriately influencing the representation or interpretation of reported research re-
sults. Any role of the funders in the design of the study; in the collection, analyses or interpretation
of data; in the writing of the manuscript, or in the decision to publish the results must be declared
in this section. If there is no role, please state “The funders had no role in the design of the study; in
the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision
to publish the results”.

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Figure captions:
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 12 April 2021 doi:10.20944/preprints202104.0311.v1

Scheme 1. (a) The schematic diagram of the atmospheric pressure microplasma setup for the
preparation of CS/RB-AgNPs solutions; (b) CS+AgNO3 solution before and after RT-APM treat-
ment and (c) the preparation of CS/RB-AgNPs/GO nanocomposite by freeze-drying process (For
interpretation of the references to color in this figure legend, the reader is referred to the web ver-
sion of this article.).
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 12 April 2021 doi:10.20944/preprints202104.0311.v1

Figure 1. (a) UV-vis spectra of all sample solutions and (b) TEM image of the RB-AgNPs with size
distribution (For interpretation of the references to color in this figure legend, the reader is re-
ferred to the web version of this article.).
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Figure 2. (a) The photographs of typical nanocomposite samples; (b) TGA curve, (c) DSC curve
obtained from the second heating, and (d) The FT-IR spectra of pure CS, CS/RB-AgNPs, and
CS/RB-AgNPs/GO nanocomposites (For interpretation of the references to color in this figure leg-
end, the reader is referred to the web version of this article.).
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Figure 3. SEM images of (a) pure CS, (b) CS/RB-AgNPs, (c) CS/RB-AgNPs/GO-0.5, and (d) mean
compressive elastic modulus of nanocomposite samples (n=3 for each group) (For interpretation of
the references to color in this figure legend, the reader is referred to the web version of this arti-
cle.).

Figure 4. (a) UV-vis spectra of GO, GO-FL, FL solutions and (b) MB, CS/RB-AgNPs-MB, CS-MB,
and CS/RB-AgNPs solutions; Inset: photos showing colours of each as-prepared solution (For in-
terpretation of the references to color in this figure legend, the reader is referred to the web ver-
sion of this article.).
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Figure 5. (a) Single drug release profile of MB; (b) single drug release profile of FL; and dual
drug release profile of (c) MB and (d) FL from CS/RB-AgNPs/GO-1.5 (For interpretation of the
references to color in this figure legend, the reader is referred to the web version of this article.).
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 12 April 2021 doi:10.20944/preprints202104.0311.v1

Figure 6. Fitting of single and dual drug release curves to Korsmeyer-Peppas model (solid lines)
(For interpretation of the references to color in this figure legend, the reader is referred to the web
version of this article.).
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Figure 7. The interaction mechanism of GO, MB and FL (For interpretation of the references to
color in this figure legend, the reader is referred to the web version of this article.).

Figure 8. Antibacterial behaviour of nanocomposite samples against S. aureus and E. coli (For inter-
pretation of the references to color in this figure legend, the reader is referred to the web version of
this article.).
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Figure 9. Photothermal conversion of all testing samples (a) Temperature profile and (b) IR ther-
mal images of PBS solution control, pure CS, CS/RB-AgNPs, CS/RB-AgNPs/GO-0.5, and CS/RB-
AgNPs/GO-1.5 under 808 nm continuous wavelength laser irradiation (1.0 W) from 0 to 3 min. (c)
Temperature profile of CS/RB-AgNPs/GO-1.5 with different power from 0.2 to 2.0 W. (d) Photo-
thermal conversion stability of CS/RB-AgNPs/GO-1.5 irradiated by 808 nm laser (power: 1.0, 1.4,
and 2.0 W). The sample was irradiated by 3 min and then cooled down to 23 °C with three repeat-
ing cycles (For interpretation of the references to color in this figure legend, the reader is referred
to the web version of this article.).

Tables:

Table 1 Nomenclatures of Samples and Their Preparation Methods.

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Sample name GO wt % (vs CS) AgNO3 / CS precursor RT-APM

concentration
Pure CS none none no
CS/RB-AgNPs none 4.0 mM AgNO3 yes
CS/RB-AgNPs/GO-0.5 0.5 % (w/w) 4.0 mM AgNO3 yes
CS/RB-AgNPs/GO-1.5 1.5 % (w/w) 4.0 mM AgNO3 yes

Table 2. The kinetic parameters in Korsmeyer-Peppas models.

Sample code Korsmeyer-Peppas model fit all range

DK (×10-5 cm2s-1) N R2
(CS/RB-AgNPs)-MB/GO-1.5 pH=7.4 1.573 0.098 0.983
(CS/RB-AgNPs)-MB/GO-1.5 pH=4.0 1.949 0.121 0.915
CS/RB-AgNPs/(GO-1.5)-FL pH=7.4 0.0231 0.155 0.981
CS/RB-AgNPs/(GO-1.5)-FL pH=4.0 0.0168 0.347 0.986
(CS/RB-AgNPs)-MB/(GO-1.5)-FL pH=7.4 MB 0.572 0.126 0.969
(CS/RB-AgNPs)-MB/(GO-1.5)-FL pH=4.0 MB 0.811 0.142 0.936
(CS/RB-AgNPs)-MB/(GO-1.5)-FL pH=7.4 FL 0.0437 0.161 0.995
(CS/RB-AgNPs)-MB/(GO-1.5)-FL pH=4.0 FL 0.0596 0.254 0.968