Photodiagnosis and Photodynamic Therapy 35 (2021) 102417

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Photodiagnosis and Photodynamic Therapy

journal homepage: www.elsevier.com/locate/pdpdt

Photo-enhanced antibacterial activity of polydopamine-curcumin

nanocomposites with excellent photodynamic and photothermal abilities
Rixiang Su a, Hongjun Yan a, Peiyuan Li a, *, Baoqu Zhang b, Ying Zhang a, Wei Su b, *
a
College of Pharmacy, Guangxi University of Chinese Medicine, Nanning, China
b
Guangxi Key Laboratory of Natural Polymer Chemistry and Physics, Nanning Normal University, Nanning 530001, P. R. China

A R T I C L E I N F O A B S T R A C T

Keywords: Background and objective Photodynamic therapy (PDT) and photothermal therapy (PTT) have gradually become
Polydopamine options for select anti-tumor and antibacterial treatment . The combination of PDT and PTT show great research
Curcumin value, which may greatly improve the curative effect. The aim of the present study was to prepare a compound
nanocomposites
system of polydopamine and curcumin (PDA-Cur nanocomposites) with excellent antibacterial effect towards
Antibacterial
Photodynamic therapy
Gram-positive and Gram-negative bacteria.
Photothermal therapy Methods Dopamine hydrochloride was oxidized and self polymerized in alkaline condition to form PDA-Cur
nanocomposites. The structure and morphology of PDA-Cur were characterized by transmission electron mi­
croscopy (TEM), scanning electron microscopy (SEM), laser scattering microscopy (LSM), ultraviolet spectro­
photometer (UV-vis), infrared spectroscopy (IR) and fluorescence emission spectrometer. Using 1,1-diphenyl-2-
picrylhydrazyl radical (DPPH), 1,3-diphenylbenzofuran (DPBF) and 2′ ,7′ -Dichlorodihydrofluorescein diacetate
(DCFH-DA) were used to detect the production of reactive oxygen species (ROS). The thermal stability of PDA-
Cur nanocomposites was investigated by temperature rising test. The antibacterial effect of PDA-Cur was
determined by plate counting technique using Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) as
models. In addition, the stability and antibacterial mechanism of PDA-Cur were investigated. Finally, the
biocompatibility was evaluated by cytotoxicity and hemolysis tests.
Results The compound system of polydopamine and curcumin was successfully prepared, which showed
improved stability compared with Cur. The consumption of DPBF by the singlet oxygen produced by PDA-Cur
was as high as 80%. In the heating test, the highest temperature increased to 59 ◦ C, which contributed to the
photodynamic and photothermal inactivation of bacteria. PDA-Cur nanocomposites showed good antibacterial
activity against S. aureus and E. coli. Under 405 nm light, the bactericidal rate of PDA-Cur against S. aureus can
reach 100% at a low concentration of 10− 4 nM, and that against E. coli was 100% at 1 nM. Under 405 + 808 nm
light, the bactericidal rate of PDA-Cur against E. coli enhanced to 100% at 0.1 nM. In addition, PDA-Cur had low
cytotoxicity and negligible hemolytic activity, showing good biocompatibility.
Conclusion PDA-Cur nanocomposites had good photodynamic effect, photo thermal conversion ability and
biocompatibility. Compared with free Cur, the antibacterial activity of PDA-Cur was significantly improved, and
the antibacterial effect with combined light was stronger than that of free Cur. Therefore, the construction of
PDA-Cur nanocomposites have confirmed that the combination of PDT and PTT can greatly improve the anti­
bacterial effect and reach bactericidal effect at low concentration, which provides a strategy for the design of
next generation antimicrobial agents.

1. Introduction
Antibiotics play an important role in the treatment of various bac­
terial infections or pathogenic microbial infections, however, the abuse
of antibiotics has become a serious problem threatening public health all
over the world [1,2]. The great harm is the generation of drug-resistant
bacteria. Therefore, it is of significance to develop new and effective
antimicrobial agents.
Photodynamic therapy (PDT) is a technology for the diagnosis and
treatment of diseases. PDT uses a specific wavelength of light to excite

* Corresponding authors.
E-mail addresses: lipearpear@163.com (P. Li), suwmail@163.com (W. Su).

https://doi.org/10.1016/j.pdpdt.2021.102417
Received 16 March 2021; Received in revised form 5 June 2021; Accepted 22 June 2021
Available online 26 June 2021
1572-1000/© 2021 Elsevier B.V. All rights reserved.
R. Su et al.
photosensitizer, which can transfer energy between photosensitizer and
oxygen, thus producing singlet oxygen (1O2) and other reactive oxygen
species (ROS) [3], inducing cell apoptosis and microbial inactivation.
Antimicrobial PDT is a method with broad antibacterial spectrum and
can kill drug-resistant strains [4-7]. Photothermal therapy (PTT) uses
photothermal conversion materials under the excitation of a specific
light source to convert light energy into heat energy, resulting in
excessive heat, leading to protein denaturation or cell membrane dam­
age of bacteria and cells, so as to achieve the purpose of sterilization. The
purpose of phototherapy is to transform bacteria into thermophilic cells
by phototherapy, which shows great potential in the treatment of
drug-resistant bacteria and bacterial biofilm [8]. The combination of
PDT and PTT has a good therapeutic effect in the treatment of tumor
[9-11], which is also an effective treatment for drug-resistant bacterial
infection [12-16]. Under the action of PDT and PTT, the inhibitory effect
of toluidine blue O and indocyanine green at high concentration on
biofilm formation and cell viability of Streptococcus mutans was signifi­
cantly enhanced [15]. The dual-mode antibacterial conjugated polymer
nanoparticles had photothermal effect and sensitization to surrounding
oxygen. Under the combined irradiation of near-infrared light and white
light, the inhibition rate of Escherichia coli was higher than that of single
PTT or PDT [16].
Curcumin (Cur) mainly comes from Curcumae Longae Rhizoma, Cur­
cumae Radix, Curcumae Rhizoma and other traditional Chinese medicine.
Because of its antibacterial, anti-inflammatory, antioxidant, anti-tumor,
anti-virus and other pharmacological effects [17-21], Cur has a good
application prospect in medical treatment [22-24], especially in the
application as antibacterial agents [25-27]. However, Cur is almost
insoluble in water with a solubility of only 11 ng / mL [28], resulting in
its low bioavailability [29] and chemical instability [30], which limits
the clinical application of Cur. Nanomaterials (1-100 nm) have been
widely used in the preparation of high-performance or multifunctional
composites due to their small volume, large specific surface area and
high surface energy. Compared with the conventional polymer com­
posite system, the physical properties, chemical properties and me­
chanical properties of nanocomposites have been improved, which can
make the organic phase and inorganic phase composite in the nano size
range [31,32]. Therefore, the unique surface structure of nanomaterials
can provide a large number of adsorption sites for the combination of
drugs and nanomaterials. The solubility and stability as well as
bioavailability of Cur can be improved by empolying nanomaterials as
carriers. The bioavailability of Cur solid lipid nanoparticles was 9.43
times higher than that of Cur suspension [33]. Moreover, a large number
of studies have indicated that Cur, which has the advantages of high
efficiency, safety, less side effects, repeated use and relatively low cost,
can be used as a photosensitizer [34,35] and possesses an excellent
antibacterial PDT effect [36,37]. For example, when Cur was encapsu­
lated in nanomofs@CMFP, the stability, antioxidant and antibacterial
activities of Cur were improved, indicating that Cur could be used as a
super long-acting food preservative with controllable release of Cur
[38]. When nano silica was employed to wrap Cur, the antibacterial
photodynamic inactivation effect was improved [39].
Nowadays, nanomaterials have attracted increasing attention in drug
delivery [40,41]. Polydopamine (PDA) is formed by oxidative self
polymerization of dopamine under alkaline conditions [42], which is
often used as a drug loaded nano material due to its good properties
[43-45]. The morphology and size of PDA can be changed by adjusting
the ratio and concentration of synthetic raw materials [46,47].
Furthermore, the strong NIR absorbance and high energy conversion
efficiency of PDA make it an excellent photothermal agent [48], and the
excessive heat generated by laser irradiation of PDA leads to cancer cell
death. In addition, PDA has good adhesion, hydrophilicity, stability and
biocompatibility [49-51]. The composite system of PDA can greatly
improve the antibacterial effect [52-54]. Under near-infrared radiation,
the MoS2/PDA-RGD modified titanium rods showed a high sterilization
rate of 94.6% against Staphylococcus aureus [55] .
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Photodiagnosis and Photodynamic Therapy 35 (2021) 102417
In this paper, PDA-Cur nanocomposites were successfully prepared,
and the singlet oxygen generation and temperature rise were detected.
The antibacterial effects of PDA-Cur on Staphylococcus aureus (S. aureus)
and Escherichia coli (E. coli) were compared between PDT alone and PDT
combined with PTT. The combination of PDT and PTT can significantly
improve the antibacterial performance, which provides a strategic
reference for the development of new antimicrobial agents.
2. Materials and Methods
2.1. Main materials
Curcumin was purchased from Aladdin reagent Co., Ltd. (Shanghai,
China). Dopamine hydrochloride was obtained from McLean Biochem­
ical Technology Co., Ltd. (Shanghai, China). Tris (hydroxymethyl)
aminothane was bought from Adamas Reagent Co., Ltd. Isopropanol and
ethanol were purchased from Xilong science and Technology Co., Ltd.
S. aureus and E. coli were obtained from Luwei Technology Co., Ltd.
(Shanghai, China). Agar and broth were bought from Luqiao Technology
Co., Ltd. (Beijing, China). Cell counting kit-8 (CCK-8) was obtained from
Beijing solarbio science technology co., ltd.
2.2. Synthesis of PDA-Cur nanocomposites
0.12 g of tris (hydroxymethyl) animomethane (Tris) was dissolved in
the mixture of ultra pure water (100 mL) and isopropanol (20 mL),
adjusted to pH = 8.5 with 1% hydrochloric acid. Then, 0.05 g of
dopamine hydrochloride was added under violent stirring, centrifuged
at 14000 rpm for 20 min, washed twice with water and ethanol water to
obtain PDA. The PDA and Cur were mixed and stirred at the same
concentration of 1:1, and then lyophilized to prepare PDA-Cur.
2.3. Characterization of PDA-Cur
The size and morphology of PDA and PDA-Cur in aqueous solution
were determined by transmission electron microscopy (TEM). A drop of
sample (100 μM) aqueous solution was deposited on a 300-mesh carbon-
coated copper mesh, dried at room temperature, and the sample was
observed using TEM. The surface morphology and size of PDA and PDA-
Cur were also observed by scanning electron microscope (SEM). The
particle size distribution of PDA and PDA-Cur were measured by laser
scattering microscope (LSM). The UV-Vis absorption spectra of PDA, Cur
and PDA-Cur were obtained with a UV-5200 PC spectrophotometer. The
infrared spectra of 4000-500 cm− 1 were analyzed by infrared spec­
trometer (NICOLET-iS10). The fluorescence intensity of the sample was
measured at 420 nm by fluorescence spectrometer (RF6000).
2.4. Stability of PDA-Cur
In order to evaluate the stability of PDA-Cur in normal environment,
pure water, 0.9% NaCl solution and phosphate buffer saline (PBS) were
used to prepare Cur and PDA-Cur solutions respectively, which were
placed in the dark at room temperature. The absorbance at 424 nm was
measured by UV-vis spectrophotometer at 0, 12, 24, 36, 48, 60 and 72h,
respectively. At the same time, the particle size of PDA-Cur solution after
7 days was measured by TEM and LSM.
2.5. Determination of 1O2
In order to explore the antibacterial mechanism, the production of
ROS including 1O2 were measured using two methods. Firstly, the
antioxidant activity of samples were test using sodium ascorbate
(vitamin C) and the colorimetric probe 1,1-diphenyl-2-picrylhydrazyl
radical (DPPH) [56]. Briefly, the sample solution (1 μM) was irradi­
ated with 405 nm laser light for 10 minutes, and mixed with sodium
ascorbate methanol solution (10 μg/mL) with equal molar ratio, and
R. Su et al. Photodiagnosis and Photodynamic Therapy 35 (2021) 102417

Fig. 1. Characterization of PDA-Cur. (a) SEM images of PDA (left) and PDA-Cur (right). (b) TEM images of PDA and PDA-Cur. (c) LSM and particle size distribution of
PDA and PDA-Cur. (d) UV-vis absorption spectra of PDA,Cur and PDA-Cur. (e) IR spectra of PDA, Cur and PDA-Cur. (f) Fluorescence emission spectra of PDA, Cur and
PDA-Cur in 0.9% NaCl solution. The excitation wavelength was 420 nm.

3
R. Su et al.
then DPPH methanol solution (0.1 mM) was added with the ratio of 1:1
by volume. After reacting in the dark for 30 min, the UV spectra were
measured. Secondly, 1,3-diphenylbenzofuran (DPBF) was used as a
capture agent to further confirm the type of ROS. 1.5 mL of PDA, Cur or
PDA-Cur sample (1 μM) was added in the cuvette, and filled with oxygen
for 10 min, respectively. Then, 36 μL of (DPBF) solution (5 × 10− 3
mol/L) was added into the sample and mixed evenly. Finally, the ab­
sorption peak of the mixture was measured at 416 nm with UV spec­
trophotometer with different time of 405 nm light irradiation.
2.6. ROS production in bacteria
ROS production in bacteria were measured by employing 2′ ,7′ -
Dichlorodihydrofluorescein diacetate (DCFH-DA) as the capture agent
[57]. DCFH-DA solution (1 mM) was dissolved in ethanol, diluted 1000
times with 0.9% NaCl solution before use. PDA-Cur solution and bac­
terial solution prepared with 0.9% NaCl solution were mixed and
incubated at 37 ℃ for 30 minutes. The light group was irradiated with
808 nm and 405 nm dual laser light simultaneously for 10 minutes. The
above mixture and DCFH-DA working solution were mixed and stained
in the dark for 30 minutes to observed the green fluorescence under
fluorescence microscope.
2.7. Test of temperature rise curve
100 μL of sample was irradiated by 808 nm laser lamp, and the
temperature rise was recorded every 30 s until the temperature did not
rise again. To investigate the repeated heating ability of the sample, the
laser lamp was turned off to let the temperature droped to the initial
temperature, then the sample was heated again. The above steps was
repeated for 3 times.
2.8. Measurement of antimicrobial activity
S. aureus and E. coli were cultivated in lysogeny broth (LB) medium
under shaking (180 rpm) at 37 ◦ C, washed twice with 0.9% NaCl solu­
tion, then diluted to OD600 = 0.5 for S. aureus or OD600 = 0.3 for E. coli,
which was resuspended in 0.9% NaCl solution. PDA, Cur and PDA-Cur
were diluted with 0.9% NaCl solution to prepare the required concen­
tration (10− 6, 10− 4, 10− 2, 1 nM and 10, 20 μM). Two experimental
groups and control groups were set for each concentration of each
sample. Bacterial suspensions (10 μL) were mixed with 90 μL of PDA,
Cur and PDA-Cur. After incubation at 37 ◦ C for 30 min, one group was
irradiated with 405 nm laser light, and the other group was irradiated
with 808 nm and 405 nm dual laser light simultaneously for 40 min,
while the control group was not exposed to light. At the end of illumi­
nation, the nutrient broth was diluted and coated on LB agar plates, and
the number of bacterial colonies was counted after incubation at 37 ◦ C
for 24 h.
2.9. SEM observation of bacterial morphology
Scanning electron microscopy (SEM) was used to monitor the
morphological changes of the bacterial surface. The samples were pre­
pared as follows: after incubation with Cur or PDA-Cur sample solutions,
respectively, with or without irradiation, the bacteria was washed with
0.9% NaCl solution for 3 times. Then, the bacteria was centrifuged at
5000 rpm for 5 min, and fixed with 2.5% glutaraldehyde for 4 h to
maintain its original shape. After dehydration with 30%, 50%, 70%,
80%, 90% and 100% ethanol, the bacteria in a drop of 100% ethanol
were deposited on a slide for SEM examination. Untreated bacteria was
used as control.
2.10. Cytotoxicity evaluation
In order to evaluate the cytotoxicity of PDA-Cur, the cell viability
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Photodiagnosis and Photodynamic Therapy 35 (2021) 102417
was determined using the CCK-8 assay. L02 cells (human normal he­
patocyte) were cultured in complete RPMI-1640 medium, supplemented
with 10% fetal bovine serum (FBS) and 100 IU/ mL of penicillin strep­
tomycin. L02 cells were inoculated into 96-well plates at the density of 5
× 104 cells per well and incubated for 24 h. Then, the cells were treated
with PDA-Cur at different concentrations (2, 4, 6, 8 and 10 μM) at 37 ◦ C
and 5% CO2 for 1, 3, 5 and 7 days, respectively. Finally, 5% CCK-8 (100
μL) was added to each well and incubated for 2 h, and the absorbance of
CCK-8 was recorded using a microplate reader (BioTek Instruments, Inc,
USA) at the wavelength of 450 nm.
2.11. Hemolysis rate test
Whole blood was collected from mice and stored in anticoagulant
tubes containing sodium citrate. RBCs were isolated from the whole
mouse blood by centrifugation at 2500 rpm for 5 min, and then resus­
pended in 0.9% NaCl solution to reach 2% RBCs suspension. The RBCs
suspension was mixed with the required sample at the ratio of 1:1, and
the experimental group, negative control group and positive control
group were set up. In the experimental group, 0.5 mL of 2% RBCs sus­
pension was mixed with 0.5 mL of PDA, Cur or PDA-Cur sample solution,
and the final concentration were 10− 4, 10− 3, 10− 2, 10− 1 and 1 μM,
respectively. The RBCs suspension incubated with ultrapure water and
0.9% NaCl solution was set as the positive control and negative control,
respectively. After incubation at 37 ◦ C for 1 h, the mixture was centri­
fuged at 4000 rpm for 5 min. The supernatant was taken and the
absorbance was measured at 545 nm by UV spectrophotometer. The
percentage of hemolysis was calculated using the following equation:
Hemolysis% = (sample absorbance - negative control absorbance) /
(positive control absorbance - negative control absorbance) × 100%
3. Results and discussion
3.1. Composite of PDA-Cur
TEM images (Fig. 1b) revealed that the PDA exhibited a relatively
uniform spherical shape, and the nanoparticles were approximately 120
nm in diameter, which was in good consistency with the diameter size
(124 nm) obtained by LSM (Fig. 1c). SEM images showed that the PDA
was round with smooth surface and uniform particle size, while PDA-
Cur were closely integrated into clusters of different sizes (Fig. 1a),
which was consistent with the diameter dimensions of PDA-Cur (40.5,
95.7, 139.5, 236.1 and 376.0 nm) measured by LSM (Fig. 1c). The
structure of PDA contains abundant π electron cloud, which can produce
π-π interaction with other molecules containing π system so that it can
adsorb small molecules [58]. Therefore, PDA could adsorb Cur to form
complexes with different sizes (Fig. 1a). In the UV-vis spectrum
(Fig. 1d), PDA showed a very wide range of absorption, from 0 to 700
nm. Cur exhibited strong characteristic absorption peaks near 264 and
424 nm, respectively, which were strong K-band absorption of benzene
ring and long-chain conjugated system [59]. There was a characteristic
peak of Cur in the UV-vis spectrum of PDA-Cur, with no obvious
displacement, which indicated that Cur had been successfully loaded on
PDA. In the IR spectra (Fig. 1e), PDA showed a wide peak at 3394 cm− 1,
which was attributed to O-H and N-H bonds in aromatic ring [60]. The
characteristic bands at 1612 and 1510 cm− 1 belonged to C=O stretching
vibration and N-H bending vibration peaks, respectively [61]. The
characteristic peak of Cur at 3504 cm− 1 was attributed to the stretching
vibration of -OH, the peak at 1508 cm− 1 was attributed to the stretching
vibration of the ketone group [62], while the characteristic bands at
1602 and 1280 cm− 1 were attributed to the stretching vibration of
benzene ring and C-O, respectively [63]. PDA-Cur showed the charac­
teristic absorption peaks of PDA and Cur, and no new absorption peaks
appeared. At an excitation wavelength of 420 nm, there was no fluo­
rescence in the aqueous solution of PDA. The Cur solution showed a high
fluorescence intensity at 560 nm, while the fluorescence intensity of
R. Su et al.
Fig. 2. Stability of PDA-Cur. Chemical stability of Cur and PDA-Cur in different time: (a) pure water (b) 0.9% NaCl solution and (c) PBS. (d) TEM images of PDA-Cur
solution after 7 days. (e) LSM and particle size distribution of PDA-Cur solution after 7 days.
PDA-Cur was lower than that of Cur (Fig. 1f). This was due to the fact
that Cur was evenly and tightly wrapped by PDA, which was conducive
to the slow release of Cur, and helpful to reduce loss and enhance sta­
bility during storage. These results indicated that PDA and PDA-Cur had
been successfully prepared.
3.2. The stability of Cur was improved by PDA
The stability of Cur loaded on nano PDA, Cur and PDA-Cur were
monitored for 72 h. As shown in Fig. 2a-2c, Cur exhibited a low stability
and was degraded in a short time in aqueous medium, with the quan­
titative degradation value of 95%, 88% and 68% in pure water, 0.9%
NaCl solution and PBS, respectively. PDA-Cur showed the degradation
rate of 31%, 35% and 32%, respectively, which demonstrated the
improved stability of PDA-Cur compared with Cur. The morphology and
particle size of the PDA-Cur solution after being placed for 7 days were
also investigated to evaluate the stability of PDA-Cur. There was no
significant change in the particle size of the PDA-Cur solution after being
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Photodiagnosis and Photodynamic Therapy 35 (2021) 102417
placed for 7 days (Fig. 2d, 2e). Because the surface modification of PDA
depends on similar catecholamine functional groups, it can form organic
nano scale films with high chemical stability on the surface of materials
[64-66]. Moreover, plentiful of aromatic rings on the surface of PDA can
make the therapeutic molecules reach a high payload [67,68], therefore,
Cur can be loaded onto the PDA layer through π− π stacking and / or
hydrogen bonding to increase its stability.
3.3. PDA-Cur enhances the generation of 1O2
The antioxidant activity of nano PDA-Cur was tested using sodium
ascorbate (vitamin C) and the colorimetric probe DPPH. When DPPH
radicals react with antioxidants, the dark purple DPPH radicals are
reduced to yellow DPPH-H molecules. The fading degree of DPPH
molecules has a quantitative relationship with the number of electrons
received, and the absorption value at 519 nm wavelength decreases.
Therefore, quantitative analysis can be carried out by the change of
absorbance. Vitamin C as an antioxidant can reduce DPPH free radicals,
R. Su et al. Photodiagnosis and Photodynamic Therapy 35 (2021) 102417

the color of the solution will change from bright purple to light yellow.
The ROS produced by PDA-Cur in solution is expected to react with
vitamin C to quench the antioxidant properties of vitamin C. As shown in
Fig. 3, when DPPH solution was added in the presence of PDA-Cur/
vitamin C mixture, the absorbance peak at 517 nm was decreased. The
persistence of purple color of DPPH means that ROS produced by PDA-
Cur could effectively eliminate the activity of antioxidant vitamin C,
which proved the production of ROS.
In PDT, a specific wavelength of excitation light was needed to excite
the photosensitizer to produce 1O2. The 405 nm laser lamp was selected
as the light source, and DPBF was employed as the 1O2 capture agent
[69,70], which can react with 1O2 quickly [71,72]. The ability of DPBF
to produce 1O2 can be detected by monitoring the UV-visible absorption
light intensity at 416 nm. As shown in Fig. 4a, with the increase of
illumination time, the absorbance of H2O was almost unchanged, while
the absorbance of Cur and PDA-Cur showed a significant downward
Fig. 3. Oxidation of vitamin C by PDA-Cur under visible light, evident in a
trend. This indicated that DPBF in the solution was continuously
DPPH colorimetric antioxidant activity assay.
consumed, demonstrating the production of 1O2. After 10s of irradiation
(Fig. 4b), the decrease rate of Cur and PDA-Cur were 50% and 80%,
respectively, while that of H2O was less than 5%, suggesting that Cur

Fig. 4. PDA-Cur enhanced singlet oxygen generation under 405 nm laser light. (a) Decomposition curve of DPBF by pure water, PDA, Cur and PDA-Cur. (b) The
absorbance drop rate graph of H2O, PDA, Cur and PDA-Cur.

6
R. Su et al. Photodiagnosis and Photodynamic Therapy 35 (2021) 102417

Fig. 5. Investigation of temperature rise curve under 405 nm laser light. (a) The temperature change diagram of PDA, Cur and PDA-Cur solution after illumination.
(b) The temperature change diagram of PDA-Cur after repeated illumination.

Fig. 6. Antibacterial activity of PDA-Cur

against S. aureus. (a) Agar plate photographs
of Cur and PDA-Cur (0.01 nM) against S. aureus
before and after light treatment, respectively.
(b) SEM images of blank S. aureus. (c) Treat­
ment of S. aureus with PDA-Cur under light
irradiation. The antibacterial activities of Cur
and PDA-Cur against S. aureus under (d) non
illumination, (e) 405 nm laser irradiation and
(f) 808 + 405 nm laser irradiation. The error
bars indicate the standard deviation of three
independent replicates.

7
R. Su et al.
could produce 1O2 under the excitation of light, and the combination of
PDA and Cur increased the production amount and rate of 1O2 and could
be used in photodynamic therapy.
3.4. Thermal stability was investigated by heating curve test
In PTT, photothermal conversion materials is employed under the
excitation of a specific light source, so that light energy changes into
heat energy and results in excessive heat. The photothermal conversion
of PDA was tested under 808 nm light source, and its thermal stability
was investigated through repeated temperature rise curve test. As shown
in Fig. 5a, the rising temperature of PDA-Cur was higher than that of
Cur, indicating that PDA-Cur had stronger photothermal conversion
effect, which was due to the excellent heating effect of PDA (Fig. 5a).
PDA could play a stronger role in light and heat conversion when Cur
was tightly wrapped in PDA. In repeated tests, the temperature of PDA-
Cur increased from 25 to 59 ◦ C (Fig. 5b), showing the stable photo­
thermal effect of PDA-Cur.
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Photodiagnosis and Photodynamic Therapy 35 (2021) 102417
Fig. 7. Antibacterial activity of PDA-Cur
against E. coli. (a) Agar plate photographs of
Cur and PDA-Cur (1nM) against E. coli before
and after light treatment, respectively. (b) SEM
images of blank E. coli. (c) Treatment of E. coli
with PDA-Cur under light irradiation. The
antibacterial activities of Cur and PDA-Cur
against E. coli were (d) non illumination; (e)
405 nm laser irradiation; (f) 808 + 405 nm laser
irradiation. The error bars indicate the standard
deviation of three independent replicates.
3.5. Photoinactivation of bacteria
3.5.1. Photoinactivation of Staphylococcus aureus
Colony counting demonstrated that Cur and PDA-Cur effectively
photo-inactivated the bacteria (Fig. 6a), while showed no obvious effect
on the survival rate of S. aureus in the dark (Fig. 6a, 6d). To confirm the
damage of bacterial cell surface, the morphological changes of bacterial
surface were examined by scanning electron microscopy (SEM). The
bacteria treated with normal saline was used as control, which had clear
edge and regular oval surface integrity in SEM images (Fig. 6b). After
treated with PDA-Cur, the bacteria was destroyed (Fig. 6c), a large
number of cytoplasmic leakage and morphological changes being
observed. Under 405 nm illumination (Fig. 6e), the bactericidal rate of
PDA-Cur reached 100% at 10− 4 nM, and the bactericidal concentration
of PDA-Cur was significantly lower than that of free Cur (20 μM).
Furthermore, the combined PDT and PTT antibacterial properties of the
drug were investigated using 808 and 405 nm dual laser lights. As shown
in Fig. 4f, the bactericidal rate of Cur was below 90% at the low con­
centration, and the antibacterial ability increased with increasing drug
concentration, which reached 100% at a concentration of 1 μM for Cur.
It worth mention that only 10− 6 nM and 10− 4 nM of PDA-Cur was
needed to eradicate 99% and 100% cells, respectively. The results
R. Su et al.
Fig. 8. ROS levels of S. aureus and E. coli under different treatments: control, Cur and PDA-Cur with or without light irradiation (0.5 mW cm− 2). Scale bar: 100 µm.
showed that the antibacterial activity of PDA-Cur against S. aureus was
better than that of Cur, and the dual lights can promote the antibacterial
ability of PDA-Cur by the combination of PDT and PTT.
3.5.2. Photoinactivation of Escherichia coli
Cur and PDA-Cur had negligible dark toxicity to E. coli under non
light conditions (Fig. 7a, 7d). SEM images showed that the morphology
of E. coli in the control group was complete and smooth (Fig. 7b), but the
surface of PDA-Cur treated E. coli exhibited some membrane defects
(Fig. 7c). Under 405 nm illumination (Fig. 7e), the bactericidal rate of
Cur was below 90% at all concentrations, and no significant change was
observed, while PDA-Cur showed 100% sterilization at 1 nM. Under the
illumination of 405 and 808 nm dual lights, there was also no significant
difference in the germicidal efficacy of Cur at different concentrations
(Fig. 7f). The germicidal efficacy increased with the increase of PDA-Cur
concentration, reaching 100% sterilization at 0.1 nM. The results
showed that the irradiation helped to improve the antibacterial ability
compared with the non light group, and the dual lights showed better
effect than single 405 nm light in the enhancement of antibacterial
ability against E. coli.
The above results showed that the antibacterial activity of Cur and
PDA-Cur towards positive S. aureus and negative E. coli under light
condition was stronger than that under non light condition, proving that
the 1O2 produced in PDT and the photothermal property of nano­
particles in PTT are important factors to improve the bactericidal rate.
Moreover, the combination of PDT and PTT showed enhanced antibac­
terial ability than singe PDT. Besides, under the dual laser lights, PDA-
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Photodiagnosis and Photodynamic Therapy 35 (2021) 102417
Cur completely killed S. aureus at 10− 4 nM (Fig. 6f), while higher con­
centration (0.1 nM) was needed to eliminate all the E. coli (Fig. 7f),
which indicated that PDA-Cur had stronger antibacterial effect on Gram-
positive bacteria than on Gram-negative bacteria.
3.6. Exploration of antibacterial mechanism
In order to verify the level of ROS in bacteria, DCFH-DA was used to
detect the change of fluorescence intensity. DCFH-DA has no fluores­
cence and can pass through the cell membrane freely. 2 ’, 7′ - dichlor­
odihydrofluorescein (DCFH) can be hydrolyzed by esterase. DCFH does
not penetrate the cell membrane, so the probe is easy to accumulate in
the cell. Reactive oxygen species (ROS) in cells could oxidize non fluo­
rescent DCFH to produce fluorescent 2 ’, 7′ - dichlorofluorescein (DCF).
The intensity of green fluorescence was proportional to the level of ROS.
As shown in Fig. 8, the bacteria in the control group and the Cur and
PDA-Cur groups without illumination only detected very weak green
fluorescence, which might be caused by the fluorescence of the bacteria
itself, so the production of ROS could be almost ignored. After irradia­
tion, green fluorescence was observed for Cur and PDA-Cur groups, and
the fluorescence of PDA-Cur group was significantly higher than that of
Cur group, indicating that PDA-Cur produced more ROS. In addition,
compared with S. aureus, the fluorescence intensity of E. coli was weaker,
which indicated that ROS was easier to enter into Gram-positive bacteria
and cause its inactivation. This was consistent with the antibacterial
results. This might be related to the inherent characteristics of cell
membrane of Gram-negative bacteria and Gram-positive bacteria,
R. Su et al.
Fig. 9. Biocompatibility evaluation of PDA-Cur. (a) Viability of L02 cells treated with different concentrations of PDA-Cur at 1, 3, 5 and 7 days. (b) Hemolysis rate of
RBCs after incubation with various concentrations of PDA, Cur and PDA-Cur. RBCs in H2O and 0.9% NaCl solution were set as positive and negative controls,
respectively. The error bars indicate the standard deviation of three independent replicates.
because Gram-negative bacteria had refractory extracellular membranes
[73,74]. These results indicated that the bactericidal activity of PDA-Cur
might be attributed to the photodynamic mechanism involving ROS. The
ROS generated in photodynamic treatment can kill the pathogen by
causing membrane and / or DNA damage [75], and photothermal action
can cause cell membrane damage [76]. The bactericidal mechanism of
PDA-Cur was to destroy the cell membrane of bacteria, and the ROS
promoted cellular content leakage (Fig. 6c) and cell membrane damage
of bacteria (Fig. 7c). Cur that was rich in C, H and O elements possessed
phenolic hydroxyl group and carbon-carbon double bond group, which
was easy to oxidize to produce ROS under laser irradiation [77],
showing strong PDT effect. PDA that was rich in C, H, O and N elements
possessed catechol, amine and imine groups, so that it had the ability to
scavenge free radicals and excellent photothermal conversion [78],
showing the effects of PDT and PTT. Therefore, the combination of PDT
and PTT made PDA-Cur show high antibacterial activity.
3.7. PDA-Cur displayed good biocompatibility
The safety of PDA-Cur nanocomposites was evaluated by cytotoxicity
measurement and hemolysis analysis. As presented in Fig. 9a, the cell
viability exhibited only a slight decrease with increasing PDA-Cur con­
centration and administration days, and the survival rate was higher
than eighty percent. Therefore, PDA-Cur had almost no cytotoxicity
towards L02 cells at 10 μM, which was much higher than its working
concentration on S. aureus (10− 4 nM) and E. coli (0.1 nM). Hemolysis test
also showed that the hemolytic activity of PDA-Cur on RBCs could be
ignored (Fig. 9b). The hemolysis rate of PDA was 3.62%, and that of
PDA-Cur was 4.02%, which was lower than that of Cur (4.93%), which
confirmed that PDA-Cur had good blood compatibility. The results
showed that PDA-Cur had good biocompatibility.
4. Conclusion
In summary,the PDA-Cur nanocomposites have been successfully
prepared with excellent photodynamic and photothermal effects.
Compared with free Cur, PDA-Cur had higher efficiency of 1O2 pro­
duction while possessed excellent transfer ability of light to heat, which
were beneficial to mediate the combined antibacterial effect of PDT and
PTT. Moreover, compared with PDT alone, the combined application of
PDT and PTT can speed up the destruction of bacterial cell membrane
and accelerate drug entry into bacteria, promoting the photodynamic
and photothermal inactivation of bacteria, and consequently possessed
significantly improved antibacterial effect of PDA-Cur on Gram-positive
bacteria and Gram-negative bacteria under the combined application of
PDT and PTT. In addition, PDA-Cur showed improved stability
10
Photodiagnosis and Photodynamic Therapy 35 (2021) 102417
compared with Cur, and its cytotoxicity and hemolytic activity were
negligible, which proved that it had good biocompatibility. This study
provides a new idea for the development of novel antibacterial agents.
Acknowledgements
This research was supported by the National Natural Science Foun­
dation of China (51961009, 21761006), Natural Science Foundation of
Guangxi Province (2018GXNSFAA281345), “BAGUI Scholar” Program
of Guangxi Province of China, Natural Science Foundation of Guangxi
University of Chinese Medicine (2017JQ001), Research Project of first-
class discipline construction of Guangxi Province (2019XK135), Project
of Guangxi Key Laboratory of Zhuang and Yao Ethnic Medicine
(GXZYZZ2019-4).
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