Synthesis and characterization of nanoprobes. The as-synthesized nanoprobes are composed of four modules, those are AuNPs, GP, diazirine and Ce6. As schematically illustrated in Fig. 2a, we firstly prepare GP-conjugated AuNPs (GP-AuNPs) through the Schiff base reaction, in which the aldehyde groups of GP (20 mg mL− 1, 100 µL) react with amino groups on AuNPs (1.5 mg mL− 1, 200 µL) surface to form Schiff base and then reduced by NaBH4 to form stable structure51. Next, we obtain Ce6-loaded GP-AuNPs (GP-AuNPs@Ce6) through electrostatic adsorption between Ce6 (1.0 mg mL− 1, 10 µL) and GP-AuNPs (1.0 mg mL− 1, 300 µL). According to the characterizations of TEM, dynamic light scattering (DLS), Zeta potential and ultraviolet (UV) (Supplementary Fig. 1), we successfully synthesize GP-AuNPs@Ce6. Finally, we modify the surfaces of GP-AuNPs@Ce6 with NHS-diazirine molecules to attain the nanoprobes (GP-dAuNPs@Ce6) through the established condensation reaction28. Upon the irradiation of 405 nm-laser, the modified diazirine are transformed into carbene moieties, which are easy to form covalent bonds among each other, leading to the aggregated products28, 57, 58. Figure 2b shows irradiation time-dependent TEM images of GP-dAuNPs@Ce6 (e.g., 405 nm, 1.0 W cm− 2). At the beginning of irradiation, we can observe spherical nanoparticles (~ 11 nm in diameter) with good dispersibility. With the increase of irradiation time, the nanoparticles gradually aggregate with each other. When the irradiation time arrives at 25 min, the aggregated nanoparticles feature a much larger diameter of ~ 150 nm. Consistent with TEM results, DLS results show that the hydrodynamic size increases from ~ 12 nm to ~ 160 nm after 25-min irradiation (Fig. 2c). Also, the surface plasmon resonance (SPR) peak of nanoparticles initially locates at ~ 520 nm while gradually shifts to a longer wavelength during irradiation (Fig. 2d). Of note, the narrow peak of SPR gradually becomes as a broad shoulder after 10-min irradiation, and the maximum absorption peak appears at ~ 700 nm after 25-min irradiation, also suggesting the formation of aggregated plasmonic nanoparticles28. Accordingly, the color of the nanoprobe solution changes from wine red (Fig. 2d-I) to bluish gray (Fig. 2d-II). In contrast, there are no obvious changes in TEM images, DLS analysis and absorption spectra of nanoprobes without photoreactive crosslinkers before and after laser irradiation (Supplementary Fig. 2). These experimental results demonstrate we can controllably aggregate the synthesized nanoprobes through photo-crosslinking reactions.
Due to the agglomeration-enhanced effects, the aggregated nanoprobes exhibit superior properties compared to their non-aggregated counterparts. As depicted in Fig. 2e, the aggregated GP-dAuNPs@Ce6 exhibits ~ 4.1-fold enhancement in fluorescence intensity compared with GP-dAuNP@Ce6 under the identical conditions. As expected, the aggregated GP-dAuNPs@Ce6 features better photothermal therapy (PTT) effects compared with GP-dAuNP@Ce6. Typically, the temperature of aggregated GP-dAuNPs@Ce6 solution rises to 52 oC under 808-nm laser irradiation for 5 min, while the temperature of GP-dAuNP@Ce6 solution only improves to 33 oC under the same conditions (Fig. 2f). In addition, the aggregated GP-dAuNPs@Ce6 displays distinct photodynamic therapy (PDT) ability, comparable to that of GP-dAuNPs@Ce6 and free Ce6 molecules at the same Ce6 concentration under 660-nm irradiation (Fig. 2g).
As we know, 405-nm light belongs to short-wavelength light and has weak penetrating ability. Notwithstanding, according to previous reports, about 40% of 405-nm laser with 1.0 W cm− 2 still penetrates the tissue when the thickness of the tissue is more than 3 mm, which is enough to trigger the aggregation of nanoparicles27,28. In this context, we first study the tissue penetration depth of the constructed probes in vitro by detecting photoacoustic signals. As shown in Fig. 2h, the photoacoustic signals of GP-dAuNPs@Ce6 with 405-nm laser irradiation (GP-dAuNPs@Ce6 (+)) gradually become weak along with the increase of chicken breast tissue thickness from 0 to 5 mm. When the thickness of chicken breast tissue even reaches to 4 mm, the photoacoustic intensity of GP-dAuNPs@Ce6 (+) is still significantly stronger than other three groups (p = 0.0007) (Fig. 2i). Such tissue penetration depth provides a guarantee for the subsequent in vivo animal experiments. These unique merits of nanoprobes lay foundation for their applications in aggregation-enhanced imaging and treatments against bacteria.
Pac-Man bacteria eat nanoprobes. Next, we confirm Pac-Man bacteria indeed eat the as-synthesized nanoprobes of GP-dAuNPs@Ce6 (Fig. 3a). In addition to TEM characterizations (Fig. 1c), we perform confocal laser scanning microscope (CLSM) experiments when Gram-positive bacteria of S. aureus (SA), M. luteus (ML), and Gram-negative bacteria of E. coli (EC), P. aeruginosa (PA) are respectively incubated with 1.0 mg mL− 1 of dAuNPs@Ce6, GP-AuNPs@Ce6 or GP-dAuNPs@Ce6 at 37 oC for 2 h, and then washed with PBS buffer. As shown in CLSM images in Fig. 3b-3e, both green fluorescence signals originated from AuNPs (first column, λex = 405 nm, λem = 500–560 nm) and red fluorescence signals originated from Ce6 (second column, λex = 405 nm, λem = 600–680 nm) can be clearly observed in all GP-AuNPs@Ce6 or GP-dAuNPs@Ce6-treated bacteria. Furthermore, green signals overlap well with red signals in the merged channel (third column), demonstrating the good co-localization between AuNPs and Ce6, and thus indicating the good stability of nanoprobes when they are incubated with bacteria. However, neither green nor red fluorescence signals are detected in dAuNPs@Ce6-treated bacteria due to the absence of GP molecules in nanoprobes for targeting bacteria (Supplementary Fig. 3). To further investigate whether Pac-Man bacteria eat nanoprobes via ABC transporter pathway, we perform inhibition assay as well as competition assay. In the inhibition assay, we can not detect the fluorescence signals of GP-dAuNPs@Ce6 in the bacteria when the bacteria are treated with the bacteria respiratory chain inhibitor (e.g., sodium azide (NaN3)) (Supplementary Fig. 4)52. In the competition assay, we observe that the fluorescence signals of GP-dAuNPs@Ce6 in bacteria gradually weaken when the bacteria are respectively incubated with GP with concentrations of 0, 20 or 100 mg mL− 1 for 5 min in advance (Supplementary Fig. 5). Both the results of inhibition assay and competition assay demonstrate the uptake mechanism of nanoprobes into bacteria is indeed through ABC transporter pathway. To further verify the specificity of synthesized nanoprobes towards bacteria over mammalian cells, COS-7 and U87MG cells are incubated with 1.0 mg mL− 1 GP-dAuNPs@Ce6 at 37 oC for 2 h, and then washed with PBS buffer. As expected, we can not observe fluorescence signals in treated COS-7 and U87MG cells (Supplementary Fig. 6), suggesting the nanoprobes are hardly internalized into mammalian cells during 2-h incubation.
It is worth pointing out that fluorescence signals of all kinds of bacteria treated with GP-dAuNPs@Ce6 after 405-nm laser irradiation for 25 min exponentially enhance (Fig. 3e) compared with those of GP-AuNPs@Ce6-treated groups without (Fig. 3b) or with laser irradiation (Fig. 3c), GP-dAuNPs@Ce6-treated groups without laser irradiation (Fig. 3d). It indicates that the GP-AuNPs@Ce6 might aggregate with the assistance of photoreactive crosslinkers. More quantitatively, the mean fluorescence intensity in GP-dAuNPs@Ce6-treated S. aureus (SA) after laser irradiation is ~ 5.2-fold stronger than that of other three groups (p < 0.0001) (Fig. 3f-i), in accordance with above CLSM imaging analysis. Similar results can be observed in other three kinds of bacteria. These results demonstrate that the nanoprobes eaten by bacteria are ready for aggregation-enhanced fluorescent imaging of diverse bacteria.
Aggregation-enhanced imaging of bacteria in superficial tissues. Next, we first prove that the proposed Pac-Man strategy enables aggregation-enhanced imaging of diverse bacteria in surface skin tissue. After the 24-h injection of 50 µL SA or PA into right or left caudal thigh of the mice, the infected mice are intravenously injected with 100 µL of 1.0 mg mL− 1 GP-dAuNPs@Ce6 (Fig. 4a) or GP-AuNPs@Ce6 (Supplementary Fig. 7a). The infected sites then would be irradiated by a laser (405 nm, 25 min), which are imaged by an in vivo optical imaging system (IVIS Lumina III) (λex = 460 nm, λem = 670 nm) at 24-h postinjection. The SA or PA concentration at the infection site during imaging is ~ 1.0 × 107 CFU, which is determined via tissue harvesting, homogenization, and culturing with CFU count53–54. As revealed in Fig. 4a, we can observe fluorescence signals at both two infected sites. Of note, the fluorescence intensity in groups treated by GP-dAuNPs@Ce6 upon laser irradiation substantially improves, ~ 2.3 fold higher than that of groups without laser irradiation (p < 0.0001). On the contrary, there are no significant changes in fluorescence intensity of infected site treated by GP-AuNPs@Ce6 with or without laser irradiation (p > 0.05) (Supplementary Fig. 7a).
After 24-h injection of 50 µL PBS buffers or bacteria mixture (PA + SA) into left or right caudal thigh of mice, the infected mice are intravenously injected with 100 µL of 1.0 mg mL− 1 GP-dAuNPs@Ce6 (Fig. 4b) or GP-AuNPs@Ce6 (Supplementary Fig. 7b) and then imaged at 24-h post-injection. The PA + SA concentration during imaging is ~ 1.0 × 107 CFU. As revealed in Fig. 4b, we can only observe distinct fluorescence signals at the (PA + SA)-infected site rather than PBS-treated site. Consistently, the fluorescence intensity in groups treated by GP-dAuNPs@Ce6 upon laser irradiation is ~ 2.2 fold higher than that of groups without laser irradiation (p < 0.0001). In contrast, there are no significant changes in fluorescence intensity of infected site treated by GP-AuNPs@Ce6 with or without laser irradiation (p > 0.05) (Supplementary Fig. 7b). Furthermore, no obvious fluorescence signals can be measured in the infected sites when they are treated with AuNPs or dAuNPs@Ce6 (Supplementary Fig. 8). Together, these results indicate the nanoprobes of GP-dAuNPs@Ce6 aggregate at the infected site after laser irradiation, greatly enhancing the fluorescence imaging performance.
To determine the detection limit of Pac-Man strategy, we image bacteria with a series of concentrations. Remarkably, we detect distinct fluorescent signals of SA (Fig. 4c) or PA (Fig. 4d) cells at concentrations as low as ~ 1.0 × 105 CFU in vivo by using GP-dAuNPs@Ce6 after 405-nm laser irradiation, which is around two orders of magnitude lower than most contrast agents (e.g., nuclease-activated probes, zinc-dipicolylamine probes, supramolecular nanoassemblies and antimicrobial peptides, etc) 59–61. Also, the fluorescence intensity of GP-dAuNPs@Ce6 groups with laser irradiation is ~ 2.2 fold higher than that without laser irradiation (p < 0.0001). Hence, Pac-Man strategy features an ultrahigh sensitivity, which should be sufficient for many in vivo scenarios.
Next, we test the feasibility of Pac-Man strategy for photoacoustic imaging of diverse bacteria in surface tissues by using a photoacoustic imaging system (Vevo®LAZR, VisualSonics, Inc., Canada) at 24-h postinjection of GP-dAuNPs@Ce6. As shown in Fig. 4e, SA or PA cells at concentrations as low as ~ 1.0 × 105 CFU generate negligible photoacoustic signals in AuNPs, dAuNPs@Ce6 and GP-AuNPs@Ce6-treated groups before and after laser irradiation, and in GP-dAuNPs@Ce6-treated groups before laser irradiation. In sharp contrast, SA or PA cells at concentrations as low as ~ 1.0 × 105 CFU generate much stronger photoacoustic signals in GP-dAuNPs@Ce6-treated groups after laser irradiation, and the signal intensity has ~ 15.2-fold enhancement compared with other groups (p < 0.0001). These results suggest Pac-Man strategy feature excellent photoacoustic imaging ability for mapping bacteria in surface tissues.
Aggregation-enhanced imaging of bacteria in tumour and gut. Next, we verify the effectiveness of Pac-Man strategy on imaging of bacteria in tumour and gut. Accordingly, we construct two different kinds of proof-of-concept models of bacteria in tumour xenografts and gastrointestinal tract. To construct the tumour xenografts model, we subcutaneously inject 100 µL of 4T1-LUC cells (~ 5 × 106 cells) into the right back region of female nude mice (6–8 weeks old). When the tumour grows to 100 mm3, we subcutaneously inject 50 µL of SA or PA into the left thigh region of mice (Fig. 5a & 5b) or respectively into both the left thigh region and the right tumour region of mice (Fig. 5c & 5d), followed by intravenous injection of 100 µL GP-dAuNPs@Ce6 (1.0 mg mL− 1). The infected sites as well as tumour sites are then imaged by an in vivo optical imaging system (λex = 460 nm, λem = 670 nm) or a photoacoustic imaging system at 24-h postinjection of GP-dAuNPs@Ce6. The bacterial cell concentration during imaging is ~ 1.0 ×107 CFU, which is within the range of certain commensal and therapeutic scenarios1, 62, 63. As revealed in Fig. 5a & 5b, we can observe fluorescence and photoacoustic signals only at the infected sites instead of tumour sites containing no bacteria, indicating the Pac-Man strategy enables the discrimination of bacteria from tumour. Expectedly, the detecting signals from the infected sites treated with 405-nm laser irradiation are significantly stronger than counterparts without 405-nm laser irradiation (e.g., ~ 1.5 (SA) and ~ 2.8 (PA)-fold enhancement in fluorescence signals, ~ 2.6 (SA) and ~ 4.0 (PA)-fold enhancement in photoacoustic signals) (p < 0.0001). As further revealed in Fig. 5c & 5d, we can observe fluorescence and photoacoustic signals simultaneously at the infected sites and the tumour sites containing bacteria. Consistently, the detecting signals from both the infected sites and the tumour sites containing bacteria treated with 405-nm laser irradiation are much stronger than counterparts without 405-nm laser irradiation (e.g., ~ 3.19 (SA, Left), ~ 4.66 (SA, Right) and ~ 2.4 (PA, Left), ~ 2.96 (PA, Right)-fold enhancement in fluorescence signals, ~ 3.08 (SA, Left), ~ 2.67 (SA, Right) and ~ 3.3 (PA, Left), ~ 4.5 (PA, Right)-fold enhancement in photoacoustic signals) (p < 0.0001). These results together prove the Pac-Man strategy allows the aggregation-enhanced imaging of diverse bacteria residing within tumour tissues.
To construct gastrointestinal tract model, the agarose gel containing E.coli (EC) is injected into the gut lumen of the female nude mice (6–8 weeks old). Afterwards, 100 µL of 1.0 mg mL− 1 GP-dAuNPs@Ce6 is intravenously injected into the mice. At 24-h postinjection of GP-dAuNPs@Ce6, the gut is then imaged by an in vivo optical imaging system (λex = 460 nm, λem = 670 nm) or a photoacoustic imaging system. The final concentration of EC is ~ 1.0 ×107 CFU during imaging. Indeed, distinct fluorescence as well as photoacoustic signals are measured in the gut containing EC (Fig. 5e). Also, the fluorescence intensity from the gut containing bacteria treated with 405-nm laser irradiation is ~ 1.6-fold stronger than that the counterparts without 405-nm laser irradiation. Consistently, the photoacoustic intensity from the gut containing bacteria treated with 405-nm laser irradiation is ~ 5.5-fold higher than that the counterparts without 405-nm laser irradiation. These data demonstrate the Pac-Man strategy could resolve the spatial distribution of bacteria within the gut.
Aggregation-enhanced therapy of bacteria in vitro. Next, we evaluate the in vitro antibacterial activity of the developed Pac-Man strategy. As expected, wrinkled or lysed EC (Fig. 6a) and SA cells (Fig. 6b) exhibit in scanning electron microscope (SEM) images when they are incubated with GP-dAuNPs@Ce6 for 2 h and then suffered with a series of laser irradiations (i.e., 405 nm, 1.0 W cm− 2, 25 min; 660 nm, 12 mW cm− 2, 5 min; 808 nm, 1.0 W cm− 2, 5 min), while the intact EC and SA cells exist in other control groups. As further revealed in agar plate experiments, small amount of bacterial colony of SA (Fig. 6c) or EC (Fig. 6d) exists in GP-dAuNPs@Ce6-treated groups after the order 405-nm (25 min), 660-nm (5min) and 808-nm (5 min) laser irradiations. During these irradiation processes, 405-nm laser leads to the aggregation of nanoprobes, 660-nm laser induces Ce6 to produce singlet oxygen (photodynamic therapy (PDT) effects)55–56, and 808-nm laser trigger aggregated AuNPs to yield thermal (photothermal therapy (PTT) effects)28. By contrast, numerous bacterial colonies are observed in other control groups. As further quantitatively analyzed in Fig. 6e-6f, GP-dAuNPs@Ce6 shows dominant in vitro antibacterial rates of ~ 94.5% to SA, ~ 97.6% to EC. These results demonstrate that Pac-Man strategy possesses an aggregation-enhanced anti-bacterial ability against both Gram-negative and Gram-positive bacteria in vitro.
To assess the antibacterial rates of the nanoprobes, we excise the infected tissues from the mice after the therapy, followed by homogenization, and culturing with CFU count.
Aggregation-enhanced therapy of bacteria in vivo. In order to evaluate the antibacterial ability of Pac-Man strategy in vivo, 50 µL of SA and PA are injected into the right thigh of the mice, respectively. Then, these infected mice are intravenously injected with 100 µL of GP-dAuNPs@Ce6 (1.0 mg/mL) respectively. The bacterial cell concentration is ~ 1.0 ×107 CFU during treatment. For systematic comparisons, these mice are then divided into six therapy groups (e.g., group 1 (G1): GP-dAuNPs@Ce6 + 660-nm laser; group 2 (G2): GP-dAuNPs@Ce6 + 808-nm laser; group 3 (G3): GP-dAuNPs@Ce6 + 660-nm laser + 808-nm laser; group 4 (G4): GP-dAuNPs@Ce6 + 405-nm laser + 660-nm laser; group 5 (G5): GP-dAuNPs@Ce6 + 405-nm laser + 808-nm laser; group 6 (G6): GP-dAuNPs@Ce6 + 405-nm laser + 660-nm laser + 808-nm laser. The representative photographs of these mice are displayed in Figs. 7a & 7e. As expected, earliest and fastest wound healing and scarring occurs in G6, which is further confirmed by the relative wound area (S/S0) in Figs. 7b & 7f. To assess the antibacterial rates of the nanoprobes, we excise the infected tissues from the mice after the therapy, followed by homogenization, and culturing with CFU count. In line with therapy results, CFU counts in G6 are significantly less than those of other 5 groups (p < 0.0001) (Figs. 7c & 7g). As a consequence, the in vivo antibacterial rates are calculated as 97.3 % against SA and 98.1% against PA. The high antibacterial rates are contributed to PDT as well as PTT effects. Afterwards, a series of staining experiments including hematoxylin-eosin staining, Masson’s trichrome and Gram-related staining of infected tissues from the six groups after therapy are performed. As manifested in Figs. 7d & 7h, compared with other groups, almost no cell necrosis (H&E) and clear tissue texture, no inflammatory factors (Massion) and no obvious bacteria (Gram) are found in G6. On the other aspect, the PTT effects are directly confirmed by an IR thermal imaging camera. As revealed in Figs. 7i & 7j, the local temperature of the infected tissues treated with GP-dAuNPs@Ce6 dramatically rises to 52°C after 808 nm-irradiation. In contrast, the local temperature of the tumour hardly changes in other control groups under the identical treatments. These results together prove that Pac-Man strategy shows an aggregation-enhanced anti-bacterial ability against both Gram-negative and Gram-positive bacteria in vivo.
Toxicity assessment. Furthermore, we test the cytotoxicity and in vivo toxicity of GP-dAuNPs@Ce6. We examine the cytotoxicity of nanoprobes via the established methyl thiazolyl tetrazolium (MTT) assays. As revealed in Supplementary Fig. 9, the cell viability of normal cells (e.g., LO2, HEK-293T and Marc-145 cells) as well as cancer cells (e.g., HeLa and MCF-7 cells) is above 80% even when they are incubated with 2.0 mg mL− 1 GP-dAuNPs@Ce6 for 24 h, suggesting the low-cytotoxicity of GP-dAuNPs@Ce6 in vitro. We examine the in vivo toxicity of nanoprobes via hematoxylin-eosin, Masson’s trichrome and Gram staining. As presented in Supplementary Fig. 10, no hydropic degeneration occurs in the heart tissues; no inflammatory infiltrates appear in the liver tissues; no hyperplasia exists in the spleen tissues; no pulmonary fibrosis is found in the lung tissues; glomerula structures are easily identified in the kidney tissues. Together, no obvious histopathological abnormalities are found in biopsy sections in all resected organs, indicating the feeble in vivo toxicity of the GP-dAuNPs@Ce6.