Home Nanotechnology Close to infrared II excitation nanoplatform for photothermal/chemodynamic/antibiotic synergistic remedy combating bacterial biofilm infections | Journal of Nanobiotechnology

Close to infrared II excitation nanoplatform for photothermal/chemodynamic/antibiotic synergistic remedy combating bacterial biofilm infections | Journal of Nanobiotechnology

Close to infrared II excitation nanoplatform for photothermal/chemodynamic/antibiotic synergistic remedy combating bacterial biofilm infections | Journal of Nanobiotechnology


Synthesis and characterization of BTFB@Fe@Van

The process used for synthesizing the multifunctional nanoplatform (BTFB@Fe@Van) is illustrated in Scheme 1. First, the NIR-II natural small molecule BTF-PBA was synthesized primarily based on our current work [34]. The BTFB@Fe@Van nanoparticles had been ready by the self-assembly of phenylboronic acid-modified NIR-II natural molecule BTF-PBA (Fig. 1a), antimicrobial drug Van, and Fe2+ ions. The dimethyl sulfoxide (DMSO) answer of BTF-PBA and Van was firstly combined with the DSPE-PEG2000 water answer due to the dynamic boronate ester bonds between the PBA teams in BTF-PBA and the vicinal diols of Van. Subsequently, the CDT catalyst Fe2+ ions had been added to the answer to acquire a inexperienced answer. This profitable binding of Fe2+ ions was because of the ample sulfur and nitrogen atoms in BTF-PBA. In the meantime, the water-soluble nanoparticles packaged with solely BTF-PBA (BTFB) or BTF-PBA and Fe2+ ions (BTFB@Fe) acted because the controls.

As proven in Fig. 1b, dynamic gentle scattering (DLS) outcomes point out that the typical hydrodynamic diameters (Dh) of BTFB, BTFB@Fe, BTFB@Van, and BTFB@Fe@Van had been roughly 123, 134, 138, and 168 nm, respectively. The marginally increased Dh of BTFB@Fe@Van than that of BTFB was because of the encapsulation of the antimicrobial drug Van and Fe2+ ions. All 4 nanoparticles exhibited a uniform spherical morphology, as noticed within the transmission electron microscopy (TEM) photographs (Fig. 1c and Extra file 1: Fig. S1). The Dh of BTFB@Fe@Van was additionally detected in a phosphate-buffered saline (PBS) answer containing 10% fetal bovine serum and Dulbecco’s Modified Eagle Medium. BTFB@Fe@Van had comparable Dh values in several options (Extra file 1: Fig. S2). Even after BTFB@Fe@Van has been saved for 4 weeks, its particle dimension remained unchanged, suggesting its good dispersion stability (Extra file 1: Fig. S2). The Zeta potentials of the BTFB nanoparticles within the answer and BTFB@Fe@Van had been measured. BTFB@Fe@Van had a unfavorable floor cost (− 4.8 mV) (Fig. 1d). Vitality-dispersive spectroscopy elemental mapping confirmed that C, O, and Fe had been uniformly distributed in BTFB@Fe@Van (Fig. 1e).

The ultraviolet–seen–near-infrared (UV–VIS–NIR) absorption spectra proven in Fig. 1f revealed that the synthesized BTFB@Fe@Van had a broad absorption vary of 700–1200 nm and that its extinction coefficient at 1064 nm was excessive at 3.19 L/g cm (Extra file 1: Fig. S3), which is in line with the findings of our earlier examine [34]. The attribute absorption of Van at 280 nm will also be noticed in Extra file 1: Fig. S4, indicating that BTFB@Fe@Van had efficiently entrapped Van. The Van-loading capability of the nanoparticles obtained by calculation was 28%. Determine 1f additionally signifies that BTFB@Fe@Van has a robust NIR-II emission from 900 to 1300 nm upon excitation at 808 nm.

Fig. 1
figure 1

a Structural system of NIR-II natural small molecule BTF-PBA. b Common hydrodynamic diameters of the synthesized BTFB, BTFB@Fe, BTFB@Van, and BTFB@Fe@Van utilizing DLS. c TEM picture of BTFB@Fe@Van. d Zeta potentials of BTFB and BTFB@Fe@Van in water. e Vitality-dispersive spectroscopy elemental mapping diagram of BTFB@Fe@Van. Scale bar: 50 nm. f Absorption and NIR-II fluorescence spectrum of BTFB@Fe@Van beneath 808 nm laser excitation. (Inset: NIR-II photographs of BTFB@Fe@Van beneath 808 nm laser excitation with a 1064 nm filter)

The NIR-II photothermal efficiency of BTFB@Fe@Van was rigorously explored contemplating its sturdy absorption at 1064 nm. As proven in Fig. 2a, the temperature of BTFB@Fe@Van elevated by 30 °C after its laser irradiation at 1064 nm (1.0 W cm−2) for five min at a focus of 0.1 mg mL−1, which was a lot increased than that of pure water whose temperature elevated solely by 7 °C beneath the identical irradiation. The temperature quickly elevated with growing BTFB@Fe@Van focus (Extra file 1: Fig. S5). The photothermal conversion effectivity of BTFB@Fe@Van obtained by calculation was 28.4%, which is increased than the photothermal conversion efficiencies of most conventional photothermal brokers (Fig. 2b). The photothermal efficiency of BTFB@Fe@Van exhibited no important modifications after 5 steady cycles of laser on/off irradiation, indicating its excessive photostability (Fig. 2c).

The Fenton response potential of Fe2+ ions can catalyze H2O2 to successfully produce cytotoxic ·OH species [35]. Methylene blue (MB), an ·OH indicator that may be degraded by ·OH, was chosen to analyze the ·OH manufacturing exercise of BTFB@Fe@Van. As proven in Fig. 2d, the MB absorption by BTFB@Fe@Van at 664 nm considerably decreased when BTFB@Fe@Van was incubated in H2O2 beneath acidic circumstances. A extra noticeable discount in MB absorption by BTFB@Fe@Van was noticed after it was irradiated with a 1064 nm laser (1.0 W cm−2), indicating that NIR-II photothermal impact can speed up the prevalence of the Fenton response. Furthermore, electron spin resonance (ESR) spectra (Fig. 2e) confirmed that BTFB@Fe@Van may catalyze ·OH manufacturing within the presence of H2O2 at pH 5.5. Moreover, with 1064 nm laser irradiation, the catalytic functionality of BTFB@Fe@Van was considerably enhanced. In abstract, these outcomes confirmed that BTFB@Fe@Van exhibited catalytic exercise for the Fenton response.

As a result of acidic microenvironment of the an infection websites, the in vitro Van launch efficiency of BTFB@Fe@Van was examined beneath completely different circumstances. As proven in Fig. 2f, a low focus of Van (lower than 10%) was launched after 4 h at a physiological pH of seven.4. In contrast, the discharge fee of Van was considerably quick, and its cumulative launch reached 30% when BTFB@Fe@Van was saved in an answer with a pH of 5.5. The cumulative launch of Van was additional elevated as much as practically 70% when the answer was irradiated with a 1064 nm laser (1.0 W cm−2). These outcomes revealed that the phenylboronate ester bonds between BTF-PBA and Van in BTFB@Fe@Van endowed it with pH- and photothermal-triggered the discharge of Van on the contaminated website.

Fig. 2
figure 2

a Photothermal heating curves of water and BTFB@Fe@Van (0.1 mg mL−1) beneath 1064 nm laser irradiation (1.0 W cm−2). b Linear correlation between the cooling time and unfavorable pure logarithm of the driving drive temperature. c Photothermal stability of BTFB@Fe@Van over 5 on/off cycles. d ·OH technology quantified from the lower in MB absorbance at 664 nm ([BTFB@Fe@Van] = 0.1 mg mL−1, [H2O2] = 0.1 mM, and [MB] = 1.0 mM) with completely different remedies [pH 7.4, pH 5.5, and pH 5.5 with 1064 nm laser irradiation (1.0 W cm−2)]. e ESR spectra of the aqueous options of BTFB@Fe@Van beneath completely different circumstances ([H2O2] = 0.1 mM, and [H2O2] = 0.1 mM with 1064 nm laser irradiation (1.0 W cm−2)). f Van launch from BTFB@Fe@Van beneath completely different environments (pH 7.4, pH 5.5, and pH 5.5 with 1064 nm laser irradiation (1.0 W cm−2)

In vitro antibacterial exercise of BTFB@Fe@Van

The in vitro antibacterial efficacy of BTFB@Fe@Van was investigated utilizing S. aureus, a significant gram-positive bacterium related to many acute and persistent infections encountered in medical follow, as a mannequin [36]. Inspired by the passable NIR-II photothermal functionality and Fenton catalytic efficiency, in addition to NIR-II PTT enhanced Van/Fe2+ launch, we first examined intracellular ·OH technology utilizing laser scanning confocal microscopy and using a fluorescent probe (2′,7′-dichlorofluorescin diacetate) because the ·OH indicator. As proven in Extra file 1: Fig. S6, no detectable inexperienced fluorescence was noticed within the PBS group. When BTFB@Fe and BTFB@Fe@Van had been utilized, shiny inexperienced fluorescence was noticed within the group, demonstrating elevated ·OH ranges in S. aureus. The stay/useless bacterial cell viability assay was carried out to judge the in vitro antibacterial efficacy of BTFB@Fe@Van. Residing and useless micro organism had been stained with SYTO 9 and propidium iodide (PI; inexperienced fluorescence for residing micro organism and crimson fluorescence for useless micro organism), respectively. The micro organism within the PBS, PBS + laser, and BTFB teams confirmed inexperienced fluorescence, indicating that all the micro organism had been alive (Fig. 3a). Nonetheless, some crimson fluorescence appeared within the Van, BTFB + laser, BTFB@Fe, and BTFB@Fe@Van teams, which was because of the harm to S. aureus micro organism brought on by Van, excessive temperature, and generated ·OH. In contrast, after remedy with BTFB@Fe@Van plus 1064 nm laser, nearly all micro organism confirmed sturdy crimson fluorescence, indicating important harm brought on by the synergistic NIR-II PTT/CDT/antibiotic remedy utilized utilizing BTFB@Fe@Van.

The antibacterial impact of BTFB@Fe@Van on S. aureus was additional evaluated utilizing a Luria-Bertani (LB) agar plate assay (Fig. 3b). Quite a few S. aureus colonies within the PBS and BTFB teams survived on the LB plates, revealing the inconspicuous bacteriostatic potential of BTFB. Subsequent to a quantitative evaluation, the calculated bacterial survival fee of the Van group (57.4% ± 2.6%) was not considerably completely different from its worth (57.7% ± 6.3%) obtained after exposing the group to 1064 nm laser, whereas the variety of S. aureus colonies in BTFB + 1064 nm laser and BTFB@Fe teams tended to lower with the corresponding bacterial survival charges standing at 47.4 ± 10.1% and 61.7 ± 7.05%, respectively, indicating various inhibitory results on the bacterial development as a result of hyperthermia and the presence of ·OH. The administration of two remedies demonstrated a therapeutic efficacy superior to that obtained utilizing a single remedy (BTFB@Fe + 1064 nm: 10.8 ± 3.6%, BTFB@Fe@Van: 33.7 ± 7.8%). Importantly, the expansion of colonies on plates handled with BTFB@Fe@Van plus 1064 nm laser was diminished and reached 1.9 ± 1.0%. The morphological modifications within the micro organism subjected to completely different remedies had been additionally studied utilizing scanning electron microscopy (SEM, Fig. 3d). The PBS, PBS + laser, and BTFB teams retained intact and easy membranes. A slight collapse and wrinkles had been noticed within the Van, Van + laser, and BTFB@Fe teams, whereas important destruction was noticed within the BTFB + laser, BTFB@Fe + laser, and BTFB@Fe@Van teams. Extreme holes and wrinkles appeared on the floor of the micro organism within the BTFB@Fe@Van + 1064 nm laser group. These outcomes demonstrated that BTFB@Fe@Van can successfully kill S. aureus by synergistic NIR-II PTT/CDT/antibiotic remedy.

Fig. 3
figure 3

a Stay/useless assay after completely different remedies. Scale bars: 100 μm. b Images of the LB agar plates of S. aureus after completely different remedies. c Bacterial viability of I: PBS, II: Van, III: BTFB, IV: BTFB@Fe, and V: BTFB@Fe@Van after completely different remedies. d SEM photographs of the micro organism after completely different remedies. Scale bars: 1 μm

In vitro antibiofilm exercise of BTFB@Fe@Van

As micro organism colonize, they produce sophisticated and dense biofilms, and routine antibiotic remedy can hardly penetrate them. Furthermore, bacterial biofilms supply sturdy resistance to host immune defenses and long-lasting antimicrobial resistance towards infections [2, 3]. Nonetheless, the rising PTT and ROS triggered by BTFB@Fe@Van have nice potential to destroy the membrane constructions of bacterial biofilms and kill the micro organism, which can improve the efficacy of various antibiotics and mix them to fight micro organism. The antibiofilm potential of BTFB@Fe@Van was first assessed utilizing crystal violet staining. As proven in Fig. 4a, b, the biofilms remained viable and structurally intact within the PBS, PBS + laser, and BTFB teams. In contrast, the biofilms within the Van, Van + laser, and BTFB@Fe teams misplaced biomass, whereas a extra seen discount was noticed within the BTFB + laser, BTFB@Fe + laser, and BTFB@Fe@Van teams. The BTFB@Fe@Van + 1064 nm laser group produced glorious ends in the biofilm inhibition take a look at, fully disrupting the tight biofilms. Furthermore, following numerous remedies, three-dimensional reconstruction of stay/useless staining was carried out through confocal laser scanning microscopy (CLSM) to find out antibacterial biofilm results on them (Fig. 4c). As with crystal violet staining, completely different levels of injury to the bacterial biofilms had been noticed when both single or a number of therapeutic strategies had been utilized. The BTFB@Fe@Van + 1064 nm laser group drastically eradicated the mature biofilms and eradicated the micro organism, demonstrating a big antibiofilm impact, which was because of the extremely energetic synergistic impact of NIR-II PTT-enhanced CDT and antibiotic remedy.

Fig. 4
figure 4

a Crystal violet staining of S. aureus biofilms after completely different remedies. b The corresponding quantified biomass of the biofilms of I: PBS, II: Van, III: BTFB, IV: BTFB@Fe, and V: BTFB@Fe@Van. c Three-dimensional CLSM photographs displaying stay/useless staining of S. aureus biofilms after completely different remedies (scale bars: 100 μm)

In vivo antibiofilm exercise of BTFB@Fe@Van

Inspired by the in vitro synergistic therapeutic outcomes, we investigated the in vivo synergic eradication of BTFB@Fe@Van within the presence of S. aureus biofilm infections. The mannequin utilizing severely contaminated mice was established to type the preliminary biofilm after 3 days of inoculation with S. aureus suspension. All contaminated wounds indicated apparent suppuration. First, BTFB@Fe @Van was intravenously injected into S. aureus biofilm-infected mice for NIR-II fluorescence imaging. As proven in Fig. 5a, the NIR-II fluorescence of BTFB@Fe@Van could possibly be noticed on the an infection website 2 h after the injection and the fluorescence attained its most worth at 12 h. Quantified NIR-II fluorescence indicators of the contaminated mouse fashions are introduced in Fig. 5b, which reveals that the NIR-II fluorescence imaging indicators on the peak time level had been roughly 5 instances stronger than these earlier than the injection. The NIR-II photothermal temperature on the in vivo an infection website was monitored utilizing an infrared digital camera (Fig. 5c). The temperature on the contaminated website quickly elevated to 54 °C in mice handled with BTFB@Fe@Van after 5 min of irradiation (1064 nm, 1.0 W cm−2), whereas solely a slight improve within the temperature (6 °C) was detected within the mice handled with PBS + 1064 nm laser irradiation (Fig. 5d), displaying the nice potential that PTT has in therapeutic purposes involving residing members.

Fig. 5
figure 5

a NIR-II fluorescence photographs and b corresponding quantified NIR-II fluorescence indicators of the contaminated mouse fashions at completely different time intervals. c Infrared photothermal photographs and d corresponding temperature variations on the contaminated websites with PBS or BTFB@Fe@Van remedy beneath 1064 nm laser irradiation (1.0 W cm−2, 5 min)

We randomly divided all S. aureus biofilm-infected mice into six teams: saline, Van, BTFB@Fe, BTFB@Fe + laser, BTFB@Fe@Van, and BTFB@Fe@Van + laser. We utilized 1064 nm laser irradiation to the contaminated areas of the BTFB@Fe + and BTFB@Fe@Van + laser teams after administering the injection. We captured the macroscopic look of the injuries inside 12 d after making use of the completely different remedies, as proven in Fig. 6b. No important impact was noticed over time within the management saline group, its pus scabs grew to become extraordinarily thick. In the meantime, the presence of subcutaneous wave movement within the group demonstrated pus accumulation, which was additionally confirmed by plate colony counting after the resection of infectious tissues (Fig. 6c). In contrast to the management group, the Van, BTFB@Fe, BTFB@Fe + laser, and BTFB@Fe@Van teams confirmed low exudation and suppuration after being handled with both a single remedy or two mixed remedies. The antibiofilm and wound therapeutic performances of the BTFB@Fe@Van + laser group had been superior to these of the opposite 5 teams. The contaminated website within the BTFB@Fe@Van + laser group appeared easy with none pustules. The pores and skin of the contaminated website began to progressively contract on the sixth day of remedy and the scar vanished on the twelfth day of remedy, displaying probably the most speedy restoration. Furthermore, to additional quantify the therapeutic results of various remedies, the contaminated tissues had been resected and the surviving bacterial numbers had been counted on the finish of the therapeutic course of utilizing plate colony counting. As proven in Fig. 6c, the variety of surviving micro organism within the BTFB@Fe@Van + 1064 nm laser group was considerably decrease than that in any of the opposite teams. The variety of bacterial colonies was diminished by 98.19% in comparison with that within the management group. As well as, the wound dimension, which was quantitatively evaluated all through the remedy interval (Fig. 6d), exhibited a constant development. Furthermore, we additionally investigated the serum stage of the vital inflammatory cytokines TNF-α and IL-6. Among the many six teams, the BTFB@Fe@Van + 1064 nm laser group had the bottom TNF-α and IL-6 expression and confirmed no important distinction even in comparison with the conventional mouse group (Fig. 6e, f). These outcomes additional demonstrated the wonderful in vivo synergistic therapeutic efficiency of BTFB@Fe@Van upon 1064 nm laser irradiation.

Fig. 6
figure 6

a Time line steering for antibiofilm therapeutic course of. b Digital images of the lesion website between 0 and 12 d after completely different remedies. c Images of the LB agar plates of S. aureus of the resected lesion website 12 d after the injection. d Quantitative evaluation of the relative wound space all through the therapeutic course of. e, f Serum ranges of cytokines IL-6 and TNF-α in I: Saline, II: Van, III: BTFB@Fe, IV: BTFB@Fe + 1064 nm laser, V: BTFB@Fe@Van, and VI: BTFB@Fe@Van + 1064 nm laser teams after completely different remedies

Moreover, irritation within the resected wounds was assessed utilizing hematoxylin and eosin (H&E) staining (Fig. 7a) and Masson staining (Fig. 7b). The histological staining outcomes confirmed a big infiltration of inflammatory cells within the saline group, whereas the inflammatory responses within the BTFB@Fe + laser and BTFB@Fe@Van teams brought on by bacterial colonization was lower than that within the saline group due to the mixed remedy it acquired. The BTFB@Fe@Van + 1064 nm laser group with its ample collagen deposition and the low diploma of neutrophil infiltration was superior to some other group, suggesting its favorable restoration potential. These outcomes of in vivo S. aureus biofilm an infection remedies collectively spotlight the effectiveness of BTFB@Fe@Van in combating bacterial biofilm infections through the trimodal synergistic mechanism of NIR-II PTT, CDT, and antibiotics.

Fig. 7
figure 7

Consultant a H&E staining and b Masson staining photographs of the wound subjected to completely different remedies. Scale bars: 200 μm

Biocompatibility of BTFB@Fe@Van

Along with verifying the antibiofilm properties of BTFB@Fe@Van, we carried out a collection of experiments to evaluate its biocompatibility. The in vivo biocompatibility of BTFB@Fe@Van was additional evaluated utilizing blood samples to research liver and kidney features. 14 d after intravenously injection, the biochemical index parameters of BTFB@Fe and BTFB@Fe@Van teams remained throughout the regular vary and weren’t considerably completely different from these of the management group (Fig. 8a). Furthermore, the histopathology photographs of the guts, liver, spleen, lung, and kidney of the BTFB@Fe and BTFB@Fe@Van teams additional supported their good compatibility with no indicators of an infection or irritation (Fig. 8b). These outcomes demonstrated that nanoplatform BTFB@Fe@Van has a excessive in vivo biocompatibility, highlighting its potential for biomedical purposes in residing members.

Fig. 8
figure 8

a Routine blood indexes of mice after completely different remedies at day 14 post-injection. b H&E staining impact of the primary organ sections of the mice on I: Saline, II: BTFB@Fe, and III: BTFB@Fe@Van 14 d after the injection. Scale bar: 100 μm



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