Fluorescence resonance energy transfer enhanced photothermal and photodynamic antibacterial therapy post a single injection

Lei Xue a, Qing Shen a, Tian Zhang a, Yibin Fan b, Xiaogang Xu c, Jinjun Shao *a, Dongliang Yang a, Wenli Zhao a, Xiaochen Dong *a and Xiaozhou Mou *d
aKey Laboratory of Flexible Electronics (KLOFE) & Institute of Advanced Materials (IAM), Nanjing Tech University (NanjingTech), Nanjing 211816, China. E-mail: iamjjshao@njtech.edu.cn; iamxcdong@njtech.edu.cn
bDepartment of Dermatology, Zhejiang Provincial People's Hospital, Affiliated People's Hospital, Hangzhou Medical College, Hangzhou 310014, China
cDepartment of Geriatrics, Zhejiang Provincial Key Lab of Geriatrics & Geriatrics Institute of Zhejiang Province, Zhejiang Hospital, Hangzhou 310013, China
dClinical Research Institute, Zhejiang Provincial People's Hospital, Affiliated People's Hospital, Hangzhou Medical College, Hangzhou 310014, China. E-mail: mouxz@zju.edu.cn

Received 27th April 2021 , Accepted 26th May 2021

First published on 2nd June 2021


Refractory MRSA infections seriously threaten human health, which compromises antibiotic efficacy. Although phototherapy has exhibited great feasibility in the fight against drug-resistant pathogens, a single-modal therapeutic agent without targeted performance has resulted in low drug utilization and obvious side effects. Herein, near-infrared (NIR) light-triggered theranostic nanoparticles (NDIA@PEG-Ce6/B NPs) were prepared by employing naphthalene diimide derivative NDIA as a photothermal agent, PEGylated chlorin e6 (PEG-Ce6) as a photosensitizer and PEGylated phenylboronic acid (PEG-B) as a bacteria-targeting agent. Upon laser irradiation, the singlet oxygen produced by Ce6 can efficiently kill bacteria through photodynamic therapy. Moreover, the fluorescence emission of Ce6 can be absorbed by the photothermal agent NDIA with a fluorescence resonance energy transfer (FRET) efficiency of 78% to reinforce the photothermal effect of NDIA, and the photothermal conversion efficiency of NDIA@PEG-Ce6/B NPs was up to 49.7%. In vitro and in vivo antibacterial experiments indicated that NDIA@PEG-Ce6/B NPs could targeted assembly and effectively destroy multidrug-resistant bacteria through synergistic photothermal and photodynamic therapy with 99.999% antibacterial efficacy under NIR light illumination. The study provides a nanosystem for the antibacterial treatment of synergistic photodynamic and photothermal therapy for subcutaneous abscesses, as well as a novel FRET strategy for further design of light-triggered antibacterial nanoplatforms.


Methicillin-resistant Staphylococcus aureus (MRSA) can cause a variety of infections that are resistant to conventional antibiotic treatment, such as skin and soft tissue infections, bacteremia, osteomyelitis, infective endocarditis, and pneumonia.1–4 Many antibiotics have been developed to fight against MRSA infections, such as penicillin, teicoplanin and linezolid.5–7 However, bacteria can promptly develop resistance to antibiotics when the antibiotics are introduced into clinical practice, bringing severe challenges in the prevention and treatment of clinical MRSA infections. Therefore, it is of great urgency to develop new effective antibacterial strategies instead of antibiotics.8–16

Recently, photothermal therapy (PTT) and photodynamic therapy (PDT), with features of noninvasiveness and high selectivity, have received ever-growing attention in the field of antibacterial treatment.17–22 Generally, PTT alone requires a high temperature of up to 70 °C to completely kill bacteria, which brings pain to patients and even damages nearby normal tissues.23,24 Lowering the temperature can reduce the side effects, however, the antibacterial efficacy may also be significantly impeded. PDT has been reported to utilize photosensitizers to produce reactive oxygen species (ROS) under photoirradiation, effectively killing Gram-negative and Gram-positive bacteria.25–27 In particular, PDT and PTT rarely result in bacterial resistance. It is anticipated that the temperature increase can enhance the permeability of photosensitizers into bacteria, so as to promote the tissue penetration of photosensitizers and further promote the photodynamic efficacy.28–30 In turn, the ROS produced by photosensitizers can reduce the heat resistance of bacteria, thereby enhancing the PTT efficacy. Combining photodynamic and photothermal therapies for synergistic antibacterial treatment is a promising method to surmount the above problems.31–34

Fluorescence resonance energy transfer (FRET) is an energy exchange process between the energy donor and energy acceptor.35,36 It takes place when the distance between two luminescent groups is close enough, usually less than 10 nm, and the overlap area of the emission spectrum of the energy donor and the absorption spectrum of the energy acceptor is greater than 30%. In recent years, multifunctional phototheranostic nanoplatforms, based on intra-capsule, intra-molecule, and intra-particle FRET mechanisms, have been constructed to improve the cancer therapeutic efficiency.37–39 For example, Zhang et al. developed a series of fluorescent dyes CX with polymethine structures. With the help of FRET, micelles containing two CX dyes showed an enhanced fluorescence intensity, which could be applied for deeper tissue fluorescence imaging.40 Peng et al. developed a new strategy (BDPCR) using the single-molecule Förster resonance energy transfer mechanism to transfer part of the fluorescence energy into heat for enhanced PDT and PTT synergistic therapy.41 In general, upon photoirradiation, photosensitizer Ce6 can emit fluorescence via radiative transitions during PDT.42 Through ingenious design, the fluorescence of Ce6 can be absorbed by the energy acceptor via the FRET pathway, further boosting the phototheranostic efficacy.

Phenylboric acid (BA), a functional unit of the boron center with two hydroxyl groups, has been widely applied in the development of biochemical sensors for saccharides owing to its high recognition and binding activity towards the diols within saccharides.43 It is reported that a BA-modified dendrimer can selectively and efficiently target Gram-positive bacteria at neutral pH.44 The targeting performance of BA on Gram-positive bacteria can make the drug specifically bind to the bacterial membrane. Thus, a BA functionalized antibacterial agent can reduce the drug dosage, enhance the bactericidal efficacy and relieve the side effects of phototherapy.

Herein, phototheranostic nanoparticles NDIA@PEG-Ce6/B NPs were developed with FRET-enhanced photothermal and photodynamic antibacterial efficacy. Naphthalene diimide derivative NDIA with intensive NIR absorption was synthesized as a photothermal agent, and it was then self-assembled with Ce6-containing PEG-Ce6 and BA-containing PEG-B to fabricate the biocompatible NDIA@PEG-Ce6/B NPs (Scheme 1). Due to the overlap between the emission of PEG-Ce6 and the absorption of NDIA, NIR NDIA takes advantage of the fluorescence of PEG-Ce6 through FRET to improve the photothermal performance, and a high photothermal conversion efficiency of 49.7% was achieved for NDIA@PEG-Ce6/B NPs. In addition to the Ce6-mediated photodynamic antimicrobial effect, NIR photoirradiation can further induce hyperthermia to improve the antimicrobial efficacy. Owing to the remarkable synergistic antibacterial effect and the targeting performance of BA, NDIA@PEG-Ce6/B NPs can eradicate bacteria efficiently after a single injection.

image file: d1qm00631b-s1.tif
Scheme 1 Illustration of the NDIA@PEG-Ce6/B NPs for FRET-enhanced photothermal and photodynamic synergistic antibacterial therapies.


Materials and characterization

Unless stated otherwise, all materials were purchased from commercial sources. PEGylated boronic acid PEG-B and PEGylated chlorin e6 PEG-Ce6 were purchased from Xi’an Ruixi Biological Technology Co., Ltd. 1H and 13C NMR spectra were obtained on a Bruker 400 MHz spectrometer at room temperature and the chemical shift (δ) relative to CDCl3 is given in ppm (1H NMR is 7.26 ppm, 13C NMR is 77.0 ppm). UV-vis-NIR absorption and fluorescence spectra were recorded on a Shimadzu UV-3600 spectrophotometer and an F-4600 spectrofluorophotometer, respectively. The data of MALDI-TOF Mass were collected using a Bruker Autoflex speed MALDI-TOF Mass spectrometer. The morphology of the nanoparticles was photographed with transmission electron microscopy (JEOL JEM-2100, Japan) and the size distribution was performed by dynamic light scattering (DLS, Nanoplus-3*). An IR thermal camera (E50, Arlington) was used to record the temperature of these solution samples for photothermal performance analysis.

Synthesis of NDIA

Synthesis of 4,9-dibromo-2,7-dioctylbenzo[lmn][3,8]phenanthroline-1,3,6,8(2H,7H)-tetraone (NDI). Under a nitrogen atmosphere, 4,9-dibromobenzo[lmn][3,8]phenanthroline-1,3,6,8(2H,7H)-tetraone (NDA) (2.150 g, 5.080 mmol), 2-ethyl hexyl amine (10 mL, 61 mmol, 12 eq.), and acetic acid (160 mL) were added to a 500 mL dry round bottom flask under stirring conditions. The reaction took about 30 min after the oil bath was heated to 130 °C. After reaction liquid cooled to room temperature, a suction filter was used to get a coarse yellow product, using silica gel column chromatography purification (PE[thin space (1/6-em)]:[thin space (1/6-em)]EA = 1[thin space (1/6-em)]:[thin space (1/6-em)]1), giving a dark blue product (0.700 g, yield 21.3%). 1H NMR (400 MHz, CDCl3) δ ppm 9.00 (s, 2H), 4.20–4.10 (m, 4H), 1.568 (s, 14H), 1.387–1.350 (m, 10H), 0.95–0.912 (m, 6H). 13C NMR (100 MHz, CDCl3) δ ppm 161.19, 139.16, 128.36, 127.73, 125.25, 124.04, 45.11, 37.72, 30.57, 28.49, 23.92, 23.05, 14.07, 10.53.
Synthesis of 4,9-bis(bis(4-(dimethylamino)phenyl)amino)-2,7-dioctylbenzo[lmn][3,8]phenanthroline-1,3,6,8(2H,7H)-tetraone (NDIA). In a 100 mL three-necked round-bottom flask containing dry toluene (30 mL), N1-(4-(dimethylamino)phenyl)-N4,N4-dimethyl benzene-1,4-diamine (0.715 g, 2.800 mmol, 4.0 eq.), sodium tert-butoxide (0.235 g, 2.450 mmol, 3.5 eq.), Pd(OAc)2 (9 mg, 0.042 mmol, 0.06 eq.), DPPF (0.466 g, 0.084 mmol, 0.12 eq.) and NDI (0.454 g, 0.700 mmol) were added under a N2 atmosphere. The reaction mixture was refluxed at 115 °C for around 24 h under a N2 atmosphere. The reaction progress was monitored using TLC analysis. Once the reaction finished, the reaction mixture was gradually brought to room temperature and then precipitated with an excess of hexane to obtain a dark green solid product. The solid was again dissolved in a minimum amount of dichloromethane and further precipitated in methanol three times. The crude product was purified by flash column chromatography (silica gel, dichloromethane) to obtain the dark green solid (0.304 g, yield: 43.5%). 1H NMR (400 MHz, chloroform-d): δ ppm 8.33 (s, 2H), 6.95–6.93 (d, J = 8.0 Hz, 8H), 6.60–6.58 (d, J = 8.0 Hz, 8H), 3.94–3.86 (m, 2H,), 3.79–3.74 (m, 2H), 2.92 (s, 24H), 1.59–1.25 (m, 30H). 13C NMR (100 MHz, chloroform-d) δ ppm 163.50, 160.16, 147.63, 124.93, 113.19, 43.49, 40.71, 37.23, 30.52, 28.61, 23.07, 14.16, 10.14. MALDI-TOF Mass (m/z): calcd for: C62H76N8O4 ([M + H]+): 996.5990, found: 997.0260.

Preparation of NDIA@PEG-Ce6/B NPs

NDIA@PEG-Ce6/B NPs were fabricated through a reprecipitation method. In detail, NIR NDIA (1 mg), PEG-Ce6 (2 mg) and PEG-B (8 mg) were dissolved in 1 mL tetrahydrofuran. The above solution was rapidly injected into DI water (10 mL) under an ultrasonic water bath and then stirred overnight at room temperature to remove the tetrahydrofuran. NDIA@PEG-Ce6/B NPs were obtained and stored at 4 °C for further application in antibacterial therapy. Additionally, NDIA@F127 NPs and NDIA@PEG-Ce6 NPs were prepared with a similar method. (NDIA@F127 NPs: 1 mg NDIA, 10 mg F127. NDIA@PEG-Ce6 NPs: 1 mg NDIA, 10 mg PEG-Ce6).

Photothermal properties and photostability of NDIA@PEG-Ce6/B NPs

In a colorimetric cuvette, 1 mL of NDIA@PEG-Ce6/B NPs (100 μg mL−1) was dispersed in water and irradiated by a NIR 808 nm laser until reaching the temperature plateau. The photothermal effect of NDIA@PEG-Ce6/B NPs was evaluated at different NDIA concentrations (40, 60, 80, and 100 μg mL−1) or laser power densities (0.4, 0.6, 0.8 and 1.0 W cm−2). NDIA@PEG-Ce6/B NPs were first irradiated by a NIR 808 nm laser for 12 min and then cooled at room temperature. The heating and cooling profiles were recorded every 30 s. The cycle of the heating and cooling processes was repeated five times, and the time–temperature curve was plotted to determine the photothermal conversion efficiency of NDIA@PEG-Ce6/B NPs.

Photodynamic effect of NDIA@PEG-Ce6/B NPs

Nanoparticle solution was stimulated by a 660 nm laser with SOSG as a singlet oxygen fluorescence probe. The fluorescence intensity of SOSG at 525 nm was recorded using a fluorescence spectrometer to evaluate the singlet oxygen production capacity of NDIA@PEG-Ce6/B NPs.

In vitro antibacterial experiments

Methicillin-resistant Staphylococcus aureus (MRSA, S. aureus ATCC43300) was used in this study. MRSA was incubated into 4 mL of Luria Bertani (LB) liquid medium at 37 °C and 200 rpm in a shaker overnight. Then the bacteria were collected, centrifuged at 3000 rpm for 10 min. After being washed with sterile PBS buffer three times to clear the LB medium, the bacteria were suspended in PBS for the next experiment.

In vitro antibacterial performance evaluation of NDIA@PEG-Ce6/B NPs

To evaluate the bactericidal performance of NDIA@PEG-Ce6/B NPs without laser irradiation, 900 μL of the bacterial solution was mixed with 100 μL of PBS or NDIA@PEG-Ce6/B NPs at a series of concentrations (0, 100, 150, and 200 μg mL−1) and co-incubated for 4 h at 37 °C without shaking and then rinsed with sterile PBS buffer three times for the plate colony counting. After being treated with an 808 nm laser (1.0 W cm−2, 10 min) and a 660 nm laser (0.2 W cm−2, 5 min), the samples were diluted to 104 CFU mL−1. At last, 100[thin space (1/6-em)]μL of the diluted bacterial solution was uniformly spread on the LB plate with three parallel samples for each group, and then the plates were placed in a 37[thin space (1/6-em)]°C incubator overnight, and then a spread plate method was used to count the viable colonies.

PTT/PDT synergistic antibacterial effects of NDIA@PEG-Ce6/B NPs

To evaluate the synergistic photothermal and photodynamic antibacterial activity of NDIA@PEG-Ce6/B NPs, six groups were established: (1) bacteria + PBS + 808 nm, (2) bacteria + PBS + 660 nm, (3) bacteria + PBS + 808 nm + 660 nm, (4) bacteria + NDIA + 808 nm, (5) bacteria + NDIA + 660 nm, (6) bacteria + NDIA + 808 nm + 660 nm. The suspension mixed bacteria (900 μL) with NDIA@PEG-Ce6/B NPs (100 μL, 100 μg mL−1) were treated with an 808 nm laser and a 660 nm laser. The number of bacterial colonies was also determined by a standard plate counting assay.

Bacterial live/dead staining and ROS detection assays

1 mL of bacterial suspension was centrifuged (5000 rpm, 5 min) and re-suspended in 100 μL of PBS. A fluorescent probe mixture containing 1 μL of green fluorescent nucleic acid SYTO 9 and 1 μL of red fluorescent nucleic acid stain PI mixture (1[thin space (1/6-em)]:[thin space (1/6-em)]1) were added to the above bacterial suspension (1 in 100 dilutions). The mixture was incubated at 37 °C in the dark for 30 min. After being washed with PBS twice and fixed by 2.5% glutaraldehyde, 5 μL of the sample was dropped onto a glass slide, which was then covered with a coverslip, sealed and inspected under a confocal scanning microscope (CLSM).

To detect the generation of ROS, the bacteria were stained with DCFH-DA and fixed with 2.5% glutaraldehyde. 5 μL of the bacterial suspension were dropped on a silicon chip and CLSM was used to monitor the fluorescence intensity of the bacteria.

Morphological analysis of bacteria

To further observe the change of bacterial morphology, about 1 × 106 CFU mL−1S. aureus cells were treated with 808 nm laser and various nano agents of NDIA@PEG-B NPs, NDIA@PEG-Ce6 NPs and NDIA@PEG-Ce6/B NPs, respectively. Then, the bacteria were washed with PBS twice and fixed by 2.5% glutaraldehyde for 2 h. Afterward, the bacteria were dehydrated with different concentrations of ethanol solution (20, 40, 60, 80, 90 and 100%). Finally, the cells were dripped onto a silicon plate and characterized using the scanning electron microscope (SEM).

Cell cytotoxicity assay

The human immortalized keratinocyte (HaCaT) cells were used to test the cytotoxicity of the NDIA@PEG-Ce6/B NPs. HaCaT cells were cultured in DMEM containing 10% fetal bovine serum (FBS) and 10 μg mL−1 gentamicin and placed in a sterile incubator at 37 °C and under 5% CO2. Then the cells were incubated with different concentrations of NDIA@PEG-Ce6/B NPs (1.5625, 6.25, 25, 100 and 400 μg mL−1). MTT assay was used to explore the cytotoxicity of the nanoparticles after incubation for 48 hours.

In vivo antibacterial experiments

Twenty adult ICR female mice (permit number: SCXK(Su) 2017-0001) were bought from the Comparative Medical Center of Yangzhou University. All in vivo antibacterial evaluation experiments were conducted under the guidance of the School of Pharmaceutical Science (Nanjing Tech University) in complement with NIH guidelines and relevant laws. To build the abscess model, MRSA (20 μL) was subcutaneously injected into the right dorsal side. One day after infection, the subcutaneous abscess-bearing mice were randomly divided into four groups (n = 5): PBS, PBS + Laser, NDIA@PEG-Ce6/B NPs, and NDIA@PEG-Ce6/B NPs + Laser (Laser refers to 808 nm + 660 nm). After the abscess was injected with PBS or NDIA@PEG-Ce6/B NPs in situ, the laser groups were exposed to an 808 nm laser (1.0 W cm−2) for 10 min. After 10 min, the mice were subsequently irradiated with a 660 nm laser (0.2 W cm−2) for 5 min.

On the 3rd and 12th day, the infectious tissues were collected for Masson and H&E staining for histological analysis. After the treatment, the tissue homogenates of the infected sites in the four groups were diluted with PBS and then smeared on the LB plate for the plate colony count.

Results and discussion

Synthesis and characterization of NDIA@PEG-Ce6/B NPs

The synthetic route of NIR NDIA is illustrated in Fig. 1a. The anhydride NDA reacted with 2-ethylhexyl amine at 135 °C for 30 min to give NDI.45,46 Later, the electron-donating unit of diaryl-amine was introduced into the electron-deficient NDI core through the palladium-catalyzed Buchwald–Hartwig coupling reaction, and the donor–acceptor–donor (D–A–D) structured NIR NDIA was obtained in a yield of 43.5%.47,48 The structure and purity of NDIA were confirmed through the characterization of 1H NMR, 13C NMR and MALDI-TOF Mass spectroscopy (Fig. S1–S5, ESI). Due to the twisted intramolecular charge transfer (TICT), D–A–D structured NIR NDIA showed weak fluorescence emission. Besides, the inhibited radiation attenuation and promoted non-radiative attenuation can improve the photothermal performance of NDIA.49,50
image file: d1qm00631b-f1.tif
Fig. 1 (a) Synthetic route of NIR NDIA. (b) Normalized UV-vis-NIR absorbance of Ce6, NDIA in DCM, and NDIA@PEG-Ce6/B NPs. (c) Size distribution of NDIA@PEG-Ce6/B NPs. (d and e) TEM image of NDIA@PEG-Ce6/B NPs (scale bar: 100 nm) and corresponding size changes for 36 days. (f) Spectral overlap between NDIA and PEG-Ce6. (g) Fluorescence emission of NDIA@PEG-Ce6/B NPs and PEG-Ce6 in water (λex = 600 nm).

The absorption spectrum of NDIA in dichloromethane showed a maximum absorption peak at 823 nm, resulting from the charge transfer of 0 → 0 transitions. The characteristic peak of Ce6 appeared at 663 nm when the nanoparticles were functionalized with PEG-Ce6 (Fig. 1b). And the absorption peak of NDIA exhibited a blue-shift to 783 nm, which may be caused by the H-aggregation between molecules. Transmission electron microscopy (TEM) indicated that NDIA@PEG-Ce6/B NPs were spherical-like in shape with an average diameter of 58.6 nm (Fig. 1c and d). The hydrodynamic diameter was 73.5 nm, which suggested that NDIA@PEG-Ce6/B NPs possessed a suitable size for passive targeting. To verify the stability of the NDIA@PEG-Ce6/B NPs in an aqueous solution, the size change was monitored over 36 days by using a DLS analyzer. The size change of NDIA@PEG-Ce6/B NPs was negligible and the polydispersity index of NDIA@PEG-Ce6/B NPs was always less than 0.2, which illustrated that NDIA@PEG-Ce6/B NPs are stable in aqueous solution (Fig. 1e). Fig. 1f shows a significant spectral overlap between NDIA and PEG-Ce6, which provides the probability for FRET as the donor–acceptor pair. As shown in Fig. 1g, NDIA@PEG-Ce6/B NPs have a weak emission, which was much lower than that of PEG-Ce6, indicating that the fluorescence of Ce6 has been quenched owing to the existence of an energy acceptor. According to the FRET formula image file: d1qm00631b-t1.tif,51 where image file: d1qm00631b-t2.tif and FD are the donor fluorescence intensities with and without an acceptor, respectively, the energy transfer efficiency was calculated to be 78%.

Photothermal properties of NDIA@PEG-Ce6/B NPs

To evaluate the photothermal effect of NDIA@PEG-Ce6/B NPs, the temperature changes for NDIA@PEG-Ce6/B NPs under 808 nm laser irradiation at different power densities and concentrations were recorded. As illustrated in Fig. 2a, it was evident that at the same concentration of 100 μg mL−1, the greater the power density, the greater the temperature variation, suggesting a power density-dependent photothermal behavior. The temperature changes of NDIA@PEG-Ce6/B NPs were also measured at different concentrations (40, 60, 80 and 100 μg mL−1) under NIR photoirradiation (808 nm, 1.0 W cm−2). NDIA@PEG-Ce6/B NPs had a concentration-dependent photothermal behavior (Fig. 2b). Additionally, the photothermal conversion efficiency of NDIA@PEG-Ce6/B NPs was quantitatively investigated. 1 mL of NDIA@PEG-Ce6/B NP (100 μg mL−1) solution was irradiated by an 808 nm laser (1.0 W cm−2) for 12.5 min, followed by 15 min cooling (Fig. 2c). The temperature of NDIA@PEG-Ce6/B NPs increased by 25.1 °C, while that of the pure water only increased by 5.4 °C. The photothermal conversion efficiency of NDIA@PEG-Ce6/B NPs was calculated to be 49.7% (Fig. 2d). Besides, the NDIA@F127 NPs were prepared for comparison. The photothermal conversion efficiency of NDIA@F127 NPs was calculated to be only 37.8%, which is lower than that of NDIA@PEG-Ce6/B NPs (Fig. S6 and S7, ESI). This is due to the fact that the FRET effect manipulates the photophysical properties of NDIA@PEG-Ce6/B NPs, giving rise to elevated photon utilization. Ce6 units in NDIA@PEG-Ce6/B NPs transfer the fluorescence to NDIA, which increases the non-radiative transitions of NDIA. This resulted in the increase of the photothermal conversion efficiency of NDIA@PEG-Ce6/B NPs as well as the accomplishment of the fluorescence reuse of Ce6, which was critically important for high-performance antibacterial treatment during PTT.52
image file: d1qm00631b-f2.tif
Fig. 2 (a) Photothermal curves of NDIA@PEG-Ce6/B NPs (100 μg mL−1) at various power densities. (b) Photothermal curves of NDIA@PEG-Ce6/B NPs at different concentrations under 808 nm photoirradiation (1.0 W cm−2). (c) Heating and cooling profiles of NDIA@PEG-Ce6/B NPs (100 μg mL−1) and water. (d) Linear time data from the cooling period versus the negative natural logarithm of driving force temperature. (e) Photothermal stability of aqueous NDIA@PEG-Ce6/B NPs (100 μg mL−1). (f) Graph of the drop point of the characteristic absorption peak of NDIA and ICG within 10 min under 808 nm photoirradiation (1.0 W cm−2).

To further investigate the photostability of NDIA@PEG-Ce6/B NPs, the clinically approved organic dye indocyanine green (ICG) was applied as the reference. UV-vis spectra of NDIA@PEG-Ce6/B NPs demonstrated no noticeable change after laser irradiation (808 nm, 1.0 W cm−2, 10 min), while the absorbance of ICG dropped down by approximately 50% after the same treatment, indicating that NDIA@PEG-Ce6/B NPs can withstand prolonged exposure (Fig. 2e). NDIA@PEG-Ce6/B NPs exhibited excellent photothermal stability even after several photothermal heating cycles, demonstrating the excellent photostability and photothermal stability (Fig. 2f and Fig. S8, ESI).

Photodynamic effect of NDIA@PEG-Ce6/B NPs

Combining PTT and photodynamic therapy (PDT) is considered to be a superior method to achieve a better phototherapeutic effect. The singlet oxygen generated from NDIA@PEG-Ce6/B NPs under 660 nm laser irradiation (60 s, 0.1 W cm−2) was detected using the singlet oxygen sensor green (SOSG) probe. The photostability and dark stability of SOSG ensured the reliability of the test (Fig. 3a and Fig. S9, ESI). The fluorescence intensity of SOSG in the presence of NDIA@PEG-Ce6/B NPs increased rapidly, while that of SOSG alone was negligible. Notably, NDIA@PEG-Ce6/B exhibited a comparable fluorescence-enhancing rate of SOSG at 525 nm to free Ce6, indicating their similar generation performance (Fig. 3b–d). In addition, DPBF was used as a probe to test the photodynamic performance of NDIA itself. It was found that the photodynamic properties were negligible under the irradiation of a 660 nm laser (Fig. S10, ESI).
image file: d1qm00631b-f3.tif
Fig. 3 UV-vis spectra of light-triggered 1O2 generation for (a) SOSG, (b) Ce6 and (c) NDIA@PEG-Ce6/B under a 660 nm laser (0.1 W cm−2) with SOSG as the probe. (d) SOSG fluorescence for 1O2 detection.

In vitro antibacterial activity assay

The high photothermal conversion efficiency and good singlet oxygen generation performance contribute to the superior efficacy in killing bacteria. To further confirm the phototherapeutic efficacy of NDIA@PEG-Ce6/B NPs, the in vitro antibacterial experiments of methicillin-resistant S. aureus (MRSA) were carried out. As shown in Fig. 4a and c, no colony was found on the agar plate when treated with NDIA@PEG-Ce6/B NPs (100 μg mL−1) and the bacterial inhibition rate reached up to 99.999%, which could not be observed in other groups without laser irradiation. In the case of MRSA treated with NDIA@PEG-Ce6/B NPs only, even with a high dose of NDIA@PEG-Ce6/B NPs, the bacteria couldn’t be killed, suggesting that NDIA@PEG-Ce6/B NPs had low dark toxicity and good biocompatibility.
image file: d1qm00631b-f4.tif
Fig. 4 (a and c) Bacterial viability ratio of MRSA receiving treatments with different concentrations of NDIA@PEG-Ce6/B NPs with and without laser irradiation and the corresponding agar plate photographs. (b and d) Bacterial survival rate and the corresponding agar plate photographs of MRSA treated with a single laser or dual lasers. (e) Confocal images of PBS, NDIA@PEG-Ce6 NPs and NDIA@PEG-Ce6/B NPs incubated with MRSA and fixed by 2.5% glutaraldehyde (scale bar: 10 μm). (e) PI and SYTO 9 staining of MRSA. Group 1–8: PBS, PBS + 808 nm, PBS + 660 nm, PBS + 808 nm + 660 nm, NDIA@PEG-Ce6/B NPs, NDIA@PEG-Ce6/B NPs + 808 nm, NDIA@PEG-Ce6/B NPs + 660 nm, NDIA@PEG-Ce6/B NPs + 808 nm + 660 nm (scale bar: 50 μm).

To explore the antibacterial effect of NDIA@PEG-Ce6/B NPs, the bacteria were divided into 6 groups and plated on LB agar plates. A prominent antibacterial activity could be observed in the group NDIA@PEG-Ce6/B NPs + 808 nm + 660 nm, which was attributed to the synergistic photothermal/photodynamic therapy (Fig. 4b and d). Propidium iodide (PI) and SYTO 9 staining assay were applied to qualitatively study the synergistic phototherapeutic effect of PDT and PTT. As shown in Fig. 4e, no matter whether with or without light, the red fluorescence is very weak in the absence of NDIA@PEG-Ce6/B NPs, proving that photoirradiation alone has no killing effect on the bacteria. No evident dead bacteria were found upon NDIA@PEG-Ce6/B NP treatments in the absence of laser irradiation. Notably, the group NDIA@PEG-Ce6/B NPs + 808 nm + 660 nm treatment offered the best antibiotic effect compared to the NDIA@PEG-Ce6/B NPs + 808 nm group or NDIA@PEG-Ce6/B NPs + 660 nm group. All these results indicated that the synergistic photothermal and photodynamic effect of NDIA@PEG-Ce6/B NPs offered a better bactericidal activity.

To investigate the targeted performance of NDIA@PEG-Ce6/B NPs, the fluorescence intensity of the bacteria was photographed with a confocal laser scanning microscope (CLSM) after incubating MRSA with PBS, NDIA@PEG-Ce6 NPs and NDIA@PEG-Ce6/B NPs. As shown in Fig. 5a, when the bacteria were incubated with PBS, there is no fluorescence on the bacteria, and the bacteria showed weak fluorescence after incubation with NDIA@PEG-Ce6 NPs. When NDIA@PEG-Ce6/B NPs were incubated with the bacteria, the fluorescence on the bacteria was significantly enhanced, indicating that the boronic acid in NDIA@PEG-Ce6/B NPs had specifically interacted with peptidoglycan on the surface of MRSA.

image file: d1qm00631b-f5.tif
Fig. 5 (a) Confocal images of PBS, NDIA@PEG-Ce6 NPs and NDIA@PEG-Ce6/B NPs incubated with MRSA and fixed by 2.5% glutaraldehyde (scale bar: 10 μm). (b) Using DCFH-DA to detect ROS with a confocal microscope (scale bar: 10 μm). (c) SEM images of MRSA after various treatments: (i) PBS, (ii) PBS + laser, (iii) NDIA@PEG-Ce6/B NPs, and (iv) NDIA@PEG-Ce6/B NPs + laser (scale bar: 1 μm).

The contributions of the potential antibacterial mechanisms of NDIA@PEG-Ce6/B NPs with a laser bactericidal system were explored. The ROS generation within bacteria was also investigated with 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) as the probe.53 After incubation with or without the NDIA@PEG-Ce6/B NPs for 4 h, MRSA was exposed to 660 nm irradiation. As shown in Fig. 5b, it was confirmed that the three groups of bacteria didn’t show any fluorescence before irradiation, meaning that almost no ROS was produced in the bacteria initially. Only in the absence of NDIA@PEG-Ce6/B NPs, the bacteria of the control group using 660 nm photoirradiation have no fluorescence, indicating that almost no ROS is produced. It is worth noting that the bacteria in the NDIA@PEG-Ce6/B NPs group showed a robust green fluorescence after 10 min of irradiation, suggesting that plenty of ROS has been produced inside the bacteria. These results indicated that the interaction of NDIA@PEG-Ce6/B NPs with the 660 nm laser can produce excellent photodynamic effects.

SEM images were taken to study the morphological changes of the bacterial cell upon treatment. As shown in Fig. 5c-i and c-ii, the surface of bacteria without nanoparticle treatment was smooth even under NIR laser irradiation. On the contrary, the surface of bacteria incubated with NDIA@PEG-Ce6/B NPs and treated with laser irradiation showed obvious wrinkling and collapse, indicating that the cell membrane was destroyed (Fig. 5c-iv). It is worth noting that a large number of NDIA@PEG-Ce6/B NPs were attached to the surface of MRSA, co-incubated with NDIA@PEG-Ce6/B NPs, owing to the targeting performance of the phenyl boric acid (Fig. 5c-iii). These results were well consistent with Fig. 5a.

In vivo antibacterial performance evaluation

To ensure the biological safety for further in vivo experiments, the dark toxicity of NDIA@PEG-Ce6/B NPs was studied through MTT cell viability assay with the HaCaT cells as the cell model. No significant cytotoxicity was observed after incubation of HaCaT cell lines with NDIA@PEG-Ce6/B NPs for 24 h even at a high concentration of 400 μg mL−1 (Fig. S11, ESI), indicating the excellent biocompatibility of NDIA@PEG-Ce6/B NPs. To further investigate the synergistic photodynamic and photothermal therapeutic efficacy of NDIA@PEG-Ce6/B NPs in vivo, twenty female mice bearing MRSA were randomly divided into the following four groups (n = 5): (1) PBS, (2) PBS + laser, (3) NDIA@PEG-Ce6/B NPs only, and (4) NDIA@PEG-Ce6/B NPs + laser. The temperature of abscess sites was monitored after administration of PBS or NDIA@PEG-Ce6/B NPs into the infected tissue and irradiated with an 808 nm laser for 5 min. The thermal images and temperature change profiles in Fig. 6a and b showed that the temperature of the NDIA@PEG-Ce6/B NP group increased from 31.5 °C to 60.4 °C, while that of the PBS group just increased by 1.5 °C, further confirming the excellent PTT performance of NDIA@PEG-Ce6/B NPs.
image file: d1qm00631b-f6.tif
Fig. 6 (a) Infrared thermal images of MRSA-infected mice after injection of PBS and NDIA@PEG-Ce6/B NPs under 808 nm photoirradiation (1.0 W cm−2). (b) Temperature changes of infected sites during photoirradiation. (c) Photographs of MRSA-infected skins. Groups 1–4: PBS, PBS + laser, NDIA@PEG-Ce6/B NPs, NDIA@PEG-Ce6/B NPs. (d) Bodyweight of mice. (e) Quantitative analysis of the surviving bacteria after 14 days of therapy. (f) The corresponding spread plate photographs from infected skin after receiving different treatments.

The infected site of mice was photographed after initiation of therapy every four days. Large pustules and redness can be seen in the infected area of the PBS group and PBS + laser group. The abscess of the group treated with NDIA@PEG-Ce6/B NPs without irradiation was similar to that of the PBS group, suggesting the negligible dark toxicity of NDIA@PEG-Ce6/B NPs. On the contrary, the abscess of the group treated with NDIA@PEG-Ce6/B NPs and irradiation was much smaller than that of the group PBS + laser, indicating the effective phototoxicity of NDIA@PEG-Ce6/B NPs. Significantly, the abscess even disappeared after treatment with NDIA@PEG-Ce6/B NPs and photoirradiation for 12 days, while notably the abscess remained in other groups (Fig. 6c). Due to the excellent antibacterial effect, it can be completely treated with a single injection. Afterward, the infected skin tissues were homogenized and standard plate counting was conducted for bacterial colony count. The CFU count for each treatment was standardized according to the proportion of the control group. As exhibited in Fig. 6e and f, the bacteria in the groups of NDIA@PEG-Ce6/B NPs plus laser were almost eliminated in contrast to the other three groups. In addition, Masson staining was used to assess collagen deposition at the infected site (Fig. 7). Tissues in the NDIA @PEG-CE6/B NPS + laser group showed a darker blue colour than the other groups. These results indicated that NDIA @PEG-CE6/B NPs possess outstanding antibacterial efficacy.

image file: d1qm00631b-f7.tif
Fig. 7 Histological analysis after 3 and 12 days of treatment (scale bar: 100 μm).

Besides, the body weight changes of the mice were recorded on the 1st, 2nd, 4th, 6th, 8th, 10th, and 12th day, and no noticeable difference in the four groups was observed, indicating the excellent biosafety of NDIA@PEG-Ce6/B NPs (Fig. 6d). Moreover, for the histopathological examination, no noticeable pathological change and inflammatory lesions were observed in the major organs (Fig. S12, ESI). The result demonstrated that NDIA@PEG-Ce6/B NPs with good biocompatibility could serve as an alternative nanomaterial for combating MRSA infections.


In summary, a nano-platform of NDIA@PEG-Ce6/B NPs with fluorescence resonance energy transfer enhanced photothermal performance combined with photodynamic antibacterial performance has been constructed. NDIA@PEG-Ce6/B NPs were prepared through the nano co-precipitation method and demonstrated good water solubility, biocompatibility, and photostability. Within NDIA@PEG-Ce6/B NPs, Ce6 was taken as an energy donor and NDIA was taken as an energy acceptor for the high-efficiency FRET with an efficiency of 78%. The photothermal conversion efficiency for NDIA@PEG-Ce6/B NPs under 808 nm laser irradiation was as high as 49.1%. The photothermal imaging results show that NDIA@PEG-Ce6/B NPs can be enriched in abscesses through PEG-B mediated bacterial targeting, reducing side effects to normal tissues. In vitro and in vivo antibacterial experiments proved that NDIA@PEG-Ce6/B NPs have high phototoxicity and low dark toxicity. The reactive oxygen species and the photothermal effect produced can disrupt cell integrity and kill the bacteria efficiently. The antibacterial effect can reach 99.999% so that synergistic treatment can eliminate the abscess. At the same time, NDIA@PEG-Ce6/B NPs have almost no side effects. Tissue section studies show that NDIA@PEG-Ce6/B NPs have good biocompatibility on normal organs and tissues.

Conflicts of interest

There are no conflicts to declare.


This work was supported by the Natural Science Foundation of Jiangsu Province (BK20200092, BK20200710, and BK20190688), the Jiangsu Province Policy Guidance Plan (BZ2019014), the Six talent peak innovation team in Jiangsu Province (TD-SWYY-009), the Natural Science Foundation of Shandong Province (ZR2020KB018), and the ‘Taishan scholars’ construction special fund of Shandong Province.

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Electronic supplementary information (ESI) available. See DOI: 10.1039/d1qm00631b

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