Bacteria-Activated Theranostic Nanoprobes against Methicillin

Mar 28, 2017 - In addition, no obvious fluorescence activation was detected for the nanoprobes after incubation with MRSA culture supernatants, indica...
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Bacteria-Activated Theranostic Nanoprobes against Methicillin-Resistant Staphylococcus aureus Infection

Zhiwei Zhao,† Rong Yan,† Xuan Yi,† Jingling Li,† Jiaming Rao,† Zhengqing Guo,† Yanmei Yang,† Weifeng Li,*,† Yong-Qiang Li,*,† and Chunying Chen‡ †

School of Radiation Medicine and Protection, Medical College of Soochow University, Collaborative Innovation Center of Radiological Medicine of Jiangsu Higher Education Institutions, Suzhou 215123, China ‡ CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology of China, Beijing 100190, China S Supporting Information *

ABSTRACT: Despite numerous advanced imaging and sterilization techniques available nowadays, the sensitive in vivo diagnosis and complete elimination of drug-resistant bacterial infections remain big challenges. Here we report a strategy to design activatable theranostic nanoprobes against methicillin-resistant Staphylococcus aureus (MRSA) infections. This probe is based on silica nanoparticles coated with vancomycin-modified polyelectrolyte-cypate complexes (SiO2-Cy-Van), which is activated by an interesting phenomenon of bacteria-responsive dissociation of the polyelectrolyte from silica nanoparticles. Due to the aggregation of hydrophobic cypate fluorophores on silica nanoparticles to induce ground-state quenching, the SiO2Cy-Van nanoprobes are nonfluorescent in aqueous environments. We demonstrate that MRSA can effectively pull out the vancomycin-modified polyelectrolyte-cypate complexes from silica nanoparticles and draw them onto their own surface, changing the state of cypate from off (aggregation) to on (disaggregation) and leading to in vitro MRSA-activated nearinfrared fluorescence (NIRF) and photothermal elimination involving bacterial cell wall and membrane disruption. In vivo experiments show that this de novo-designed nanoprobe can selectively enable rapid (4 h postinjection) NIRF imaging with high sensitivity (105 colony-forming units) and efficient photothermal therapy (PTT) of MRSA infections in mice. Remarkably, the SiO2-Cy-Van nanoprobes can also afford a long-term tracking (16 days) of the development of MRSA infections, allowing real-time estimation of bacterial load in infected tissues and further providing a possible way to monitor the efficacy of antimicrobial treatment. The strategy of bacteria-activated polyelectrolyte dissociation from nanoparticles proposed in this work could also be used as a general method for the design and fabrication of bacteriaresponsive functional nanomaterials that offer possibilities to combat drug-resistant bacterial infections. KEYWORDS: bacterial infections, superbug, bioimaging, photothermal therapy, theranostic probes sensitivity by eliminating background fluorescence interference and providing a huge signal-to-noise ratio in the specific region of interest.9−14 Several exquisite microbe-activated fluorescent probes that enable sensitive in vivo diagnosis of bacterial infections have been developed, by taking advantage of the photophysical phenomenon of fluorophores’ excited-state quenching through fluorescence resonance energy transfer (FRET).13,14 However, these aforementioned probes are

B

acterial infections possess mounting public health concerns and cause an enormous medical and financial burden due to the rapid emergence of drug-resistant bacteria.1−4 Methicillin-resistant Staphylococcus aureus (MRSA), one of the main dreaded clinical pathogens (superbugs) that cause life-threatening diseases such as sepsis and acute endocarditis, accounts for numerous cases of morbidity and mortality clinically.5−8 The risk of MRSA infections can be greatly reduced if they can be sensitively diagnosed in vivo and treated at the early stage. Optical imaging based on activatable fluorescent probes is emerging as a powerful method for in vivo diagnosis, since it allows noninvasive imaging with high © 2017 American Chemical Society

Received: January 3, 2017 Accepted: March 28, 2017 Published: March 28, 2017 4428

DOI: 10.1021/acsnano.7b00041 ACS Nano 2017, 11, 4428−4438

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Figure 1. (a) Computer simulation of the interaction between bacteria and the PAH molecules absorbed on silica nanoparticles. One representative trajectory for PAH dissociation from the silica nanoparticle surface to the bacterial membrane and the time evolution of contact numbers of PAH with silica and membrane atoms are given, respectively. (b) Preparation and conceptual illustration of the SiO2-CyVan nanoprobes for MRSA-activated NIRF imaging and PTT.

typically bacterial enzyme (e.g., β-lactamase, micrococcal nuclease)-dependent and usually require complicated chemical design and synthesis processes for the preparation of the enzyme-cleavable linker molecules that connect the fluorophores and quenchers. Notably, the total number of bacterial enzyme candidates that can be employed is few, which thus becomes a major hindrance for their applications clinically. Consequently, there is an urgent need for the development of an alternative strategy to achieve bacteria-activated fluorescence that can overcome the restraints of an enzyme-dependent method. In addition, multifunctional theranostic probes that combine both bacteria-activated imaging and treatment capabilities are highly required practically for the purpose of precise and effective therapy through the concept of imagingguided treatment of bacterial infections. By controlling fluorophores’ aggregation/disaggregation on nanoparticles to induce ground-state quenching/fluorescence recovery, activatable fluorescent nanoprobes have been developed to image cellular processes, providing insight into the design of a bacteria-activated fluorescence probe.15−19 The major challenge is how to control the aggregation/disaggregation of fluorophores in a bacteria-responsive way. Layer-bylayer assembly of polyelectrolytes on nanoparticles is a commonly used strategy in nanomedicine, and many bacteriatargeted and responsive nanosystems have been reported based on polyelectrolyte-modified nanoparticles.20−23 In our prelimi-

nary computer simulations of poly(allylamine)-coated silica nanoparticles (SiO2/PAH) interacting with bacteria, it is found that PAH adsorbates are responsive to bacteria located nearby that transfer from the surface of silica nanoparticles to bacteria (Figure 1a and Figure S1). This translocation process can happen at room temperature, which points to the fact that the dissociation energy barrier of PAH from silica nanoparticle is effectively lowered by bacteria to less than 12.4 kJ/mol (typically an energy barrier of around 12.4 kJ/mol is considered to be low enough for thermodynamic translocation to happen at 300 K).24−26 Inspired by this preliminary result, bacteriaactivated fluorescence is highly expected to be achieved by exploiting the bacteria-responsive dissociation of PAHfluorophores from silica nanoparticles. Meanwhile, it has been previously reported that vancomycin (Van) exhibits a higher binding affinity with MRSA (37.2 kJ/mol) due to the formation of intimate hydrogen-bonding networks between Van and the cell wall of MRSA bacteria.27,28 Therefore, it is highly anticipated that a strategy for MRSA-activated fluorescent probe design can be developed, by combining surface chemical Van modification with bacteria-responsive SiO2/PAH-fluorophore dissociation nanosystems. Herein, we present activatable theranostic nanoprobes for sensitive near-infrared fluorescence (NIRF) imaging and photothermal therapy (PTT) of MRSA infections, based on SiO2/PAH-cypate nanosystems modified with polyethylene 4429

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Figure 2. (a) TEM image of SiO2-Cy-Van. (b) Absorption spectra of SiO2-Cy-Van and cypate. (c) Zeta potentials of SiO2 NPs, SiO2/PAH, SiO2-Cy, and SiO2-Cy-Van water solutions (pH 6.4). (d) Hydrodynamic diameter of SiO2-Cy-Van in PBS buffer (0.01 M, pH 7.4) during 7 days of storage. (e) Viability of NIH-3T3 cells after incubation with SiO2-Cy-Van at various Cy concentrations for 24 h. (f) Photothermal heating curves of SiO2-Cy-Van at various Cy concentrations under NIR laser (808 nm, 1.5 W/cm2) irradiation for 5 min.

were sequentially conjugated with the amino group of PAH through a chemical covalent coupling method (Figure 1b and Figure S2).34,35 The transmission electron microscopy (TEM) image shows that the prepared SiO2-Cy-Van nanoprobes had a regular and well-defined spherical structure with an average size of 72.7 ± 3.2 nm (Figure 2a). From the UV−vis absorption spectrum of SiO2-Cy-Van, it was found that the nanoprobes exhibited the characteristic absorption peaks of Cy at around 700 and 800 nm, indicating the successful conjugation of Cy (Figure 2b). In addition, the time-of-flight mass spectrum of SiO2-Cy-Van nanoprobes showed the characteristic peaks of Van, confirming the successful linkage of Van (Figure S3). The loading amounts of Cy and Van on SiO2-Cy-Van nanoprobes (one nanoprobe loaded with 618.15 Cy and 3.97 Van molecules) were calculated based on the standard absorption curves of Cy and Van molecules (for details, please refer to the Methods and Experiments section, Figures S4 and S5). The whole preparation process of SiO2-Cy-Van nanoprobes could also be monitored and confirmed by the reversed zeta potential and increased hydrodynamic size results (as depicted in Figure 2c and Figure S6). The prepared SiO2-Cy-Van nanoprobes demonstrated high structural stability and outstanding solubility in aqueous environments, as no significant increase of hydrodynamic size was detected for the nanoprobes during 7 days of storage in phosphate-buffered saline (PBS) buffer (Figure 2d). The methyl thiazolyl tetrazolium (MTT) assay

glycol and Van-conjugated poly(acrylic acid) molecules (PAAPEG-Van) (as depicted in Figure 1b). Cypate (Cy), a U.S. Food and Drug Administration (FDA)-approved cyanine dye, exhibits distinctive properties of NIRF emission (around 825 nm) and outstanding PTT.29−31 The PAA-PEG-Van modification is applied to confer the MRSA-responsive capability, as well as enhanced in vivo biocompatibility and relatively prolonged blood circulation.32,33 Normally, the obtained nanoprobes (SiO2-Cy-Van) are nonfluorescent in aqueous environments due to the aggregation of hydrophobic Cy fluorophores on silica nanoparticles to induce ground-state quenching.18,19 Yet in the presence of MRSA bacteria, the interactions between Van and MRSA will be stronger than the binding between PAH and silica nanoparticles. Therefore, upon the attraction of MRSA, the polyelectrolyte layers (PAH-Cy/ PAA-PEG-Van) would be partly pulled out from the silica nanoparticles and drawn onto the surface of the bacteria, changing the state of Cy from off (aggregation) to on (disaggregation) and leading to MRSA-activated NIRF imaging and PTT upon laser irradiation (Figure 1b).

RESULTS AND DISCUSSION The SiO2-Cy-Van nanoprobes were prepared by first coating negatively charged silica nanoparticles with the cationic PAH polyelectrolyte, and then Cy and the PAA-PEG-Van molecules 4430

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Figure 3. (a) Evaluation of fluorescence activation of SiO2-Cy-Van nanoprobes after incubation with bacteria for 4 h. MRSA and E. coli cultures with a concentration of 107 CFU were used, and controls included MRSA culture supernatant and the nanoprobes only. MRSA culture supernatant was obtained by centrifuging the MRSA culture to remove the MRSA bacterial cells inside. (b) Corresponding fluorescence signal intensities of the images shown in (a). *** indicates P < 0.001. (c) Representative SEM and CLSM images of MRSA (107 CFU) after incubation with Cy5.5-labeled SiO2-Cy-Van nanoprobes for 4 h and purification through low-speed centrifugation (3000 rpm). (d) Representative SEM and overlapping fluorescence images for a live/dead bacterial staining assay of MRSA (107 CFU) incubated with SiO2Cy-Van nanoprobes before and after NIR laser (808 nm, 1.5 W/cm2) irradiation for 5 min. Two fluorescent dyes were used in live/dead bacterial staining in which SYTO 9, with a green color, labeled both live and dead bacteria, while propidium iodide, with a red color, stained only dead bacteria. In the above experiments, the final concentration of SiO2-Cy-Van nanoprobes in the mixtures was 5.45 mg/mL (10 μg/mL of Cy and 0.14 μg/mL of Van).

nonfluorescent in the absence of bacteria, and activated fluorescence was found after incubation with MRSA cultures (107 colony-forming units (CFU)) for 4 h. In addition, no obvious fluorescence activation was detected for the nanoprobes after incubation with MRSA culture supernatants, indicating that the fluorescence of SiO2-Cy-Van nanoprobes was activated by bacteria rather than bacterial metabolites. Furthermore, distinct from SiO2-Cy, which is responsive to both MRSA and Gram-negative Escherichia coli (E. coli) (Figure S10), the fluorescence of SiO2-Cy-Van nanoprobes was specifically activated by MRSA, indicating the indispensability of Van for the MRSA-targeted capability of the SiO2-Cy-Van nanoprobes. The corresponding fluorescence signal intensities of the images shown in Figure 3a were quantitatively assessed. As summarized in Figure 3b and Figure S11, the Cy fluorescence in the SiO2-Cy-Van nanoprobes was not fully recovered compared to pure Cy at the same concentrations. This phenomenon indicates that only part of the polyelectrolyte layers had dissociated from the silica nanoparticles upon MRSA activation. Furthermore, the loading amount of Cy on SiO2-Cy-Van nanoprobes was found to critically affect the MRSA-activated fluorescence. For the abnormal SiO2-Cy-Van nanoprobes loaded with a higher amount of Cy (one nanoprobe loaded with 1048.98 Cy molecules rather than the normal 618.15 Cy molecules), no fluorescence enhancement was observed upon MRSA activation (Figure S12). In addition, in vitro NIRF imaging of SiO2-Cy-Van nanoprobes after 4 h of incubation with normal mammalian cells (107 of NIH-3T3 cells) was conducted, and no fluorescence enhancement was

was employed to evaluate the biocompatibility of SiO2-Cy-Van nanoprobes in vitro. As shown in Figure 2e, high cell viability (>90%) was found for the mouse embryonic fibroblast cells (NIH-3T3) after incubation with SiO2-Cy-Van nanoprobes at various Cy concentrations ranging from 1.25 to 20 μg/mL, indicating the good biocompatibility of SiO2-Cy-Van nanoprobes. The antibiotic activity of SiO2-Cy-Van nanoprobes was further tested due to the conjugation of Van, and their minimum inhibitory concentration (MIC) toward MRSA was determined to be 90 mg/mL (165 μg/mL of Cy, 2.25 μg/mL of Van) (Figure S7). Moreover, the photothermal behaviors of the SiO2-Cy-Van nanoprobes in aqueous solution under NIR laser irradiation were investigated to evaluate their photothermal performance. As shown in Figure 2f, the SiO2-Cy-Van nanoprobe solution exhibited an obvious Cy concentrationdependent photothermal effect, and a remarkable temperature increase (ΔT = 23 °C) was achieved in 300 s even at a low concentration of 5 μg/mL Cy, paving their way toward subsequent PTT of MRSA infections. Furthermore, the photothermal stability of SiO2-Cy-Van nanoprobes was investigated, and the results are shown in Figures S8 and S9. The SiO2-Cy-Van nanoprobes exhibited significantly enhanced photothermal stability over free Cy molecules under NIR laser irradiation, indicating that as the nanocarrier of Cy, the silica nanoparticles can efficiently prevent the photobleaching of Cy under NIR laser irradiation. To evaluate the MRSA-activated fluorescence of the SiO2Cy-Van nanoprobes, in vitro NIRF imaging of the nanoprobes before and after incubation with MRSA was conducted. As shown in Figure 3a, the SiO2-Cy-Van nanoprobes were 4431

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Figure 4. (a) NIRF imaging of MRSA (107 CFU)-infected mice injected with SiO2-Cy-Van nanoprobes at different time points postinjection. (b) Long-term in vivo NIRF imaging of MRSA (107 CFU)-infected mice during 20 days after SiO2-Cy-Van nanoprobe injection. (c) Average fluorescence intensities of the infection site and control site (PBS) of MRSA (107 CFU)-infected mice during 20 days after SiO2-Cy-Van nanoprobe injection. In the above experiments, the injected amount of SiO2-Cy-Van nanoprobes for MRSA-infected mice (∼20 g) was about 81.82 mg (loaded with 150 μg of Cy and 2.05 μg of Van).

only bacteria with destroyed structures of the cell wall and membrane (Figure 3d).4 As the concentration of SiO2-Cy-Van nanoprobes used here (5.45 mg/mL) is much lower than their MIC (90 mg/mL), the exhibited antimicrobial activity of SiO2Cy-Van nanoprobes is mainly attributed to their photothermal effect, showing great potential for PTT of MRSA infections. To demonstrate the performance of our nanoprobes for diagnosis of MRSA infections in vivo, an infected mouse model was first constructed by subcutaneously injecting 107 CFU of MRSA bacteria into the right caudal thigh of the mouse, while the left caudal thigh was injected with PBS as a control. The SiO2-Cy-Van nanoprobes were then intravenously injected into the infected mice through the tail vein, and dynamic in vivo NIRF imaging was conducted. As shown in Figure 4a, timedependent fluorescence activation was detected only at the infected site but was absent in the control site. Notably, bacterial infections could be clearly and rapidly imaged as early as 4 h postinjection, indicating the capability of our SiO2-CyVan nanoprobes for early diagnosis of MRSA infections in vivo. Remarkably, long-term imaging (up to 16 days) of MRSA infections was successfully achieved, indicating the robust ability of the dissociated polyelectrolyte layer (PAH-Cy/PAAPEG-Van) with the enhanced accumulation and long retention at the infected sites (Figure 4b). Compared to conventional Cybased probes, which are quickly degraded and eliminated in vivo,36 the enhanced accumulation and prolonged retention of the PAH-Cy/PAA-PEG-Van at the infected sites may result from the synergic effect of polyelectrolytes’ protection to improve Cy stability and Van modification to confer active MRSA-targeting capability.29,37−39 In addition, lipoproteins or albumin may bind to the dissociated PAH-Cy/PAA-PEG-Van layers in vivo according to a previous report,40 delaying the metabolic speed of PAH-Cy/PAA-PEG-Van in vivo and further prolonging their retention time at the infected sites. From the corresponding time-dependent fluorescence intensity profiles in Figure 4c and Figure S15, it was found that the fluorescence intensities at the infected sites increased gradually and reached

found, suggesting that normal mammalian cells do not interfere with the MRSA-activated fluorescence (Figure S13). To further confirm the polyelectrolyte dissociation-based mechanism behind the MRSA-activated fluorescence of our nanoprobes, the interaction between MRSA and SiO2-Cy-Van nanoprobes was subsequently investigated by scanning electronic microscopy (SEM) and confocal laser scanning microscopy (CLSM). As shown in Figure 3c, most of MRSA bacteria exhibited bright fluorescence from the CLSM image of the MRSA sample after incubation with SiO2-Cy-Van nanoprobes and purification through low-speed centrifugation, but only a few probe nanoparticles (indicated by red arrows) were observed on the surface of MRSA from the corresponding SEM image. These inconsistent results clearly indicated the dissociation of the polyelectrolyte layer (PAH-Cy/PAA-PEGVan) from the silica nanoparticles and subsequent absorption onto the bacterial surface. This conclusion could also be supported by the UV−vis absorption spectrum of MRSA after incubation with SiO2-Cy-Van nanoprobes and purification, in which the typical absorption peak of isolated Cy was clearly found (Figure S14). In order to evaluate the in vitro photothermal antimicrobial activity of the SiO2-Cy-Van nanoprobes, a SEM-based bacterial morphology study and a live/ dead bacterial staining assay were conducted, and the results are summarized in Figure 3d. After incubation with SiO2-Cy-Van nanoprobes but prior to laser irradiation, the MRSA cells uniformly showed clear edges and smooth bodies. On the contrary, lysed bacterial morphologies and debris were found after treatment with laser irradiation. These results clearly indicated that the SiO2-Cy-Van nanoprobes exhibit outstanding photothermal antimicrobial activity that induces significant disruptions of the bacterial cell wall and membrane. In the corresponding live/dead bacterial staining assay, MRSA cells incubated with SiO2-Cy-Van nanoprobes were stained red after laser irradiation, further confirming the disruption of the bacterial cell wall and membrane, since the red-fluorescent propidium iodide dye used here is well-known to penetrate 4432

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Figure 5. (a) In vivo NIRF imaging of MRSA (105 to 107 CFU)-infected mice injected with SiO2-Cy-Van nanoprobes at 4 h postinjection. (b) Average fluorescence intensities of the infection site and control site of MRSA (105 to 107 CFU)-infected mice injected with SiO2-Cy-Van nanoprobes at 4 h postinjection. ***P < 0.001. (c) Ex vivo tissue NIRF imaging of MRSA (107 CFU)-infected mice treated with SiO2-Cy-Van nanoprobes at 6 h postinjection. First row (from left to right): heart, liver, spleen; second row (from left to right): lung, kidney, infected tissue. In the above in vivo experiments, the injected amount of SiO2-Cy-Van nanoprobes was about 81.82 mg (loaded with 150 μg of Cy and 2.05 μg of Van).

Furthermore, NIRF imaging of ex vivo tissues from the infected mice were conducted. Since the SiO2-Cy-Van nanoprobes are nonfluorescent, the NIRF of ex vivo tissues arise from the dissociated polyelectrolyte layers (PAH-Cy/PAA-PEG-Van). As shown in Figure 5c, besides high accumulation at the infected sites, comparable bright fluorescence of Cy was also found in the kidney, indicating the kidney-based metabolic pathway of the dissociated polyelectrolyte layers. Since Van is known to be nephrotoxic, the accumulation of the dissociated PAH-Cy/PAA-PEG-Van and its toxic side effects to the kidney were subsequently investigated. As shown in Figure S19, no obvious Cy absorption peaks were found in the UV−vis absorption spectrum of the collected kidney tissues from MRSA-infected mice treated with SiO2-Cy-Van nanoprobes at 30 days postinjection, indicating that the accumulated PAHCy/PAA-PEG-Van in the kidneys could be efficiently cleared over time in vivo. In addition, compared with normal mice, no obvious kidney damage was observed from the hematoxylin and eosin (H&E) staining image of the kidney biopsy section from MRSA-infected mice treated with SiO2-Cy-Van nanoprobes at 30 days postinjection, indicating that the nanoprobes had no untoward effects on the kidney (Figure S20). For the remaining silica nanoparticles after the dissociation of polyelectrolyte layers, they were speculated to possess the liver-based metabolic pathway like other nanoparticles with a similar size reported previously.48,49 To demonstrate the performance of our nanoprobes for in vivo PTT of MRSA infections, the SiO2-Cy-Van nanoprobes were injected into mice infected by MRSA (107 CFU, subcutaneous infection model), and their PTT efficacy upon NIR laser irradiation at 4 h postinjection was assessed. Figure 6a shows representative photographs of a MRSA-infected mouse within 12 days postinjection in different treatment groups (PBS, PBS/irradiation, SiO2-Cy-Van, SiO2-Cy-Van/ irradiation), and the corresponding graphical representations of the quantitative measurement of infected wound areas are presented in Figure 6b. Although ulcerations and infected wounds were observed in all four treatment groups, the mice treated with SiO2-Cy-Van nanoprobes and laser irradiation displayed alleviated infection with earlier wound scarring and faster healing speed compared to the other three groups. Complete re-epithelialization of the infected wound was only

a maximum at 4 days postinjection, then declined gradually to the same level as the control sites, showing a synchronous trend with the evolution of MRSA cell number in the infected wound. Compared with the available bacteria-targeted fluorescent probes reported,12−14,41−47 the striking rapid and long-term tracking ability of our SiO2-Cy-Van nanoprobes allows the realtime estimation of bacterial load in infected tissues and further provides a possible way to monitor the efficacy of antimicrobial therapy. Due to the robust MRSA-activated NIRF capability to avoid background fluorescence interference, our SiO2-Cy-Van nanoprobes have the potentiality to image MRSA infections with high sensitivity. To demonstrate this, bacterial infections induced by various concentrations of MRSA bacteria ranging from 105 to 107 CFU were imaged by the SiO2-Cy-Van nanoprobes at 4 h postinjection to investigate the detection limit of our nanoprobes. As shown in Figure 5a and b, bacterial infection induced by 105 CFU of MRSA showing no skin infection symptoms (e.g., erythema and ulceration), a preclinical model of MRSA infectious disease, could be imaged and discriminated with a target-to-control ratio of 3.5. To the best of our knowledge, this is the best detection limit obtained for in vivo MRSA detection, indicating great potential for highsensitivity early diagnosis of MRSA infections in clinical settings. It should be noted that the detection limit of SiO2Cy-Van nanoprobes obtained in the current experiments is only suitable for the disease of MRSA wound infection, and the limits of detection and clinical disease are quite different for MRSA in urine or blood. In control experiments, the imaging tests of infections induced by 107 CFU of E. coli and methicillin-sensitive S. aureus (MSSA) failed with the SiO2Cy-Van nanoprobes (Figures S16 and S17), indicating the high specificity of our SiO2-Cy-Van nanoprobes for MRSA. In addition, the mice model of bacterial myositis was also created, by intramuscularly injecting MRSA (107 CFU) into the left leg muscle of mice, to investigate the performance of SiO2-Cy-Van nanoprobes for the diagnosis of MRSA infections in deeper tissues. As shown in Figure S18, NIRF imaging of MRSA myositis with SiO2-Cy-Van nanoprobes was successfully achieved at 4 h postinjection. These results reveal the capability of our nanoprobes for early diagnosis of different kinds of MRSA infections (on the skin surface or deeper sites). 4433

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Figure 6. (a) Representative photographs of the MRSA (107 CFU)-infected mice within 12 days postinjection in four different treatment groups (PBS, PBS/irradiation, SiO2-Cy-Van, SiO2-Cy-Van/irradiation). The injected amount of SiO2-Cy-Van nanoprobes was about 81.82 mg (loaded with 150 μg of Cy and 2.05 μg of Van), and NIR laser irradiation (808 nm, 1.5 W/cm2, 5 min) was employed. (b) Corresponding areas of infected wound of the mice shown in (a).

system or treated with a similar amount of free Van (2.05 μg) as used in the SiO2-Cy-Van nanoprobes was examined. As shown in Figures S21 and S22, the infected wounds in these two control samples all took almost 18 days to heal, which was much slower that the group treated with SiO2-Cy-Van nanoprobes and irradiation (12 days), indicating the advantage and great potential of our nanoprobes for in vivo PTT of MRSA infections.

found for the group treated with nanoprobes and irradiation at the 12th day postinjection. Furthermore, MRSA bacteria from the infected skin tissues at the 12th day postinjection were counted by the standard bacterial culture method to evaluate the actual efficacy of infection treatment. As shown in Figure 7a, no bacterial colony was found for the group treated with SiO2-Cy-Van nanoprobes and laser irradiation, indicating the complete recovery from infection. By contrast, bacterial growth with no significant difference in bacterial count was observed in the other three groups, revealing the poor inhibition of infections. Moreover, histological analysis in the MRSAinfected skin tissue was performed by H&E staining. As can be seen in Figure 7b, the skin biopsy section treated with SiO2Cy-Van nanoprobes and laser irradiation at the 12th day postinjection showed normal morphological features with blood vessels and hair follicles, whereas the skin in the other three groups exhibited obvious bursting and destroyed morphological features. In addition, the control sample of MRSA infection in mice recovered by their own immune

CONCLUSION In conclusion, by combining surface chemical Van modification and bacteria-responsive SiO2/polyelectrolyte-Cy dissociation nanosystems, activatable theranostic nanoprobes against MRSA infections are developed. The nanoprobes are nonfluorescent in aqueous environments because of the aggregation of hydrophobic Cy dye on the silica nanoparticles. We demonstrate that the presence of MRSA can selectively pull out the Vanmodified polyelectrolytes-Cy complexes from silica nanoparticles and draw them onto their own surface, changing the 4434

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Figure 7. (a) Photographs of bacterial cultures from the skin tissues of MRSA (107 CFU)-infected mice in four treatment groups (PBS, PBS/ irradiation, SiO2-Cy-Van, SiO2-Cy-Van/irradiation). The injected amount of SiO2-Cy-Van nanoprobes for MRSA-infected mice was about 81.82 mg (loaded with 150 μg of Cy and 2.05 μg of Van), and NIR laser irradiation (808 nm, 1.5 W/cm2, 5 min) was employed. (b) Corresponding histological images of the infected skin tissues of mice in the four treatment groups. 10 mL of 1 mM NaCl solution; then 1 mL of as-prepared silica nanoparticles was added and stirred vigorously for 3 h. The PAHcoated SiO2 NPs (SiO2/PAH) were finally collected by centrifugation at 9500 rpm for 25 min and dispersed in 1 mL of DI water. The SiO2Cy was prepared by covalently conjugating Cy with the amino group of PAH through the EDC/NHS-based covalent coupling method. First, EDC (0.5 mg, 0.0026 mmol) and NHS (0.6 mg, 0.0028 mmol) were added to 3 mL of Cy dimethylformamide (DMF) solution (0.33 mg/mL, 0.0014 mmol) and stirred for 30 min. Then, the mixture was added to 1 mL of as-obtained SiO2/PAH solution and stirred for 16 h. Finally, the SiO2-Cy was collected by centrifugation at 9500 rpm for 25 min and dispersed in DI water for further characterization and application. The number of Cy loaded on SiO2/PAH nanoparticles could be easily regulated by controlling the concentration of Cy used during SiO2-Cy nanoprobe preparation. Preparation of SiO2-Cy-Van nanoprobes. Before the preparation of SiO2-Cy-Van nanoprobes, the vancomycin and polyethylene glycol-modified poly(acrylic acid) polyelectrolyte (PAA-PEG-Van) was synthesized in advance. First, 228.6 μL of PAA (1.25 g/mL, 35 wt %) was dissolved in 1770 μL of DI water; then EDC (0.2045 g, 1.07 mmol) and NHS (0.2316 g, 1.07 mmol) were added and stirred for 30 min. Next, the mixture was added to 25 mL of mPEG-NH2 (20 mg/ mL, 0.1 mmol) and vancomycin hydrochloride (0.67 mg/mL, 0.011 mmol) solution and stirred for 24 h. The PAA-PEG-Van was then obtained by dialysis and lyophilization. The SiO2-Cy-Van nanoprobes were prepared by covalently conjugating the PAA-PEG-Van with asobtained SiO2-Cy nanoprobes. Briefly, EDC (0.3680 g, 1.92 mmol) and NHS (0.4168 g, 1.92 mmol) were added to 1 mL of PAA-PEGVan solution (50 mg/mL) and stirred for 30 min. Then, 2 mL of asprepared SiO2-Cy was added and stirred for 24 h. Finally, the SiO2-CyVan nanoprobes were collected by centrifugation at 9500 rpm for 25 min and dispersed in DI water and PBS, respectively, for further characterization and application. The amount of Cy loaded on SiO2-Cy-Van nanoprobes (MCy) can be calculated based on the equation MCy = MCy1 − MCy2. In this equation, “MCy1” represents the amount of Cy used for conjugation with the SiO2/PAH, and “MCy2” represents the amount of Cy in the supernatant after conjugation and centrifugation to remove the Cyconjugated silica nanoparticles (SiO2-Cy). The value of “MCy2” can be calculated based on the standard absorption curve of Cy (Figure S4b) after diluting the absorption value of supernatant into the range of 0.15−0.95. Similarly, the amount of Van loaded on SiO2-Cy-Van nanoprobes (MVan) can be calculated based on the equation MVan = MVan1 − MVan2. In this equation, “MVan1” represents the amount of Van in the PAA-PEG-Van molecules used for conjugation with the SiO2-

state of Cy from off (aggregation) to on (disaggregation) and leading to MRSA-targeted NIRF and photothermal elimination. Notably, rapid NIRF imaging with high sensitivity (105 CFU) and efficient imaging-guided PTT of MRSA infections are achieved in vivo based on our nanoprobes, showing great potential in early diagnosis and treatment of MRSA infections in clinical settings. Additionally, our nanoprobes can also afford long-term tracking along with the development of MRSA infections, allowing real-time estimation of bacterial load in infected tissues and further providing a possible way to monitor the efficacy of antimicrobial therapy. We believe that the concept of bacteria-responsive polyelectrolyte dissociation from silica nanoparticles could be utilized as a general but robust approach to guide the design and fabrication of bacteriaresponsive multifunctional nanomaterials and constitute their ultimate functions in the treatment of drug-resistant bacterial infections.

METHODS AND EXPERIMENTS Materials. Tetraethyl orthosilicate (TEOS), poly(allylamine hydrochloride) (PAH; Mw = 15 000 Da), poly(acrylic acid, sodium salt) (PAA; Mw = 15 000 Da), N-(3-(dimethylamino)propyl-N′-ethylcarbodiimide) hydrochloride (EDC), N-hydroxysulfosuccinimide sodium salt (NHS), vancomycin hydrochloride, and 3-(4,5-dimethyl2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) were purchased from Sigma-Aldrich. Amine-terminated PEG (mPEG-NH2; Mw = 5000 Da) was purchased from Bomei Biotechnology Company (Zhejiang, China). Cy dyes were synthesized in our own laboratory. All other chemicals were obtained from Adamas-beta and used without further purification. Deionized (DI) water (Millipore Milli-Q grade, 18.2 MΩ) was used in all the experiments. Preparation of SiO2-Cy. Silica nanoparticles (SiO2 NPs) were first synthesized according to the method reported with slight modification.50,51 Briefly, 900 μL of ammonia (28−30%) was added to a mixture containing ethanol and DI water (50 mL, Vethanol:VDI water = 4:1) and stirred for 5 min. Then, 600 μL of TEOS (0.93 g/mL) was added and stirred vigorously for 1 h, and 600 μL of TEOS was further added and stirred for 24 h. After centrifugation at 9500 rpm for 25 min and washing with ethanol and DI water three times, SiO2 NPs were dispersed in DI water for subsequent PAH coating. The PAH coating of SiO2 NPs was done based on the procedures published previously.52,53 First, PAH (100 mg, 0.0067 mmol) was dissolved in 4435

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ACS Nano Cy, and “MVan2” represents the amount of Van in the PAA-PEG-Van molecules in the supernatant after conjugation and centrifugation to remove the obtained SiO2-Cy-Van. As shown in Figure S5a, the PAA− PEG-Van molecules showed a similar absorption spectrum to that of free Van; thus the values of “MVan1” and “MVan2” can be calculated based on the standard absorption curve of Van (Figure S5b). On the basis of the procedures described above, for 90 mg/mL of normal SiO2-Cy-Van nanoprobes, the binding concentrations of Cy and Van are about 165 and 2.25 μg/mL, respectively. Moreover, on the basis of the density of bulk silica (ρ, 2.2 g/mL) and the size of the SiO2-CyVan nanoprobes (D, ∼7 × 10−6 cm), the number of SiO2-Cy-Van nanoparticles per g (N) is calculated to be 2.53 × 1015 by using the 1 equation N = . Subsequently, on the basis of the value of “N” 4π D 3 ρ*

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the propidium iodide dye (red color) penetrated only bacteria with destroyed structures of the cell wall and membrane. SEM-Based Morphology Study of Bacteria. The morphology of bacteria incubated with SiO2-Cy-Van nanoprobes before and after NIR laser irradiation was examined by field-emission scanning electron microscopy (FESEM). First, bacteria suspensions incubated with SiO2Cy-Van nanoprobes before and after NIR laser irradiation were dropped onto silicon wafers, and the silicon wafers were then treated with glutaraldehyde (2%, Sigma-Aldrich) fixation for 2 h at room temperature and ethanol gradient dehydration (50%, 70%, 90%, and 100%). Finally, the silicon wafers were completely dried by nitrogen and imaged with FESEM (S-4700, Hitachi) after platinum sputter coating. In Vivo NIRF Imaging of Bacterial Infections. Female Balb/c mice (6 weeks, ∼20 g) were purchased from Nanjing Peng Sheng Biological Technology Co. Ltd. and allowed to acclimatize for 7 days in the lab. All animal experiments were carried out in compliance with the protocols approved by the Soochow University Laboratory Animal Center. For in vivo NIRF imaging, a mice model of bacterial infection was created by subcutaneously injecting 50 μL of bacteria PBS suspension (MRSA, E. coli or S. aureus) into the right caudal thigh of Balb/c mice (injection depth: 5 mm), while the left caudal thigh was injected with PBS as a control. At 16 h postinfection, the infected mice (n = 3) were injected with 500 μL of SiO2-Cy-Van nanoprobe PBS solutions (163.64 mg/mL loaded with 150 μg of Cy and 2.05 μg of Van) through the tail vein. The infected mice were then anesthetized with 1% pelltobarbitalum natricum, and NIRF images were obtained by using an IVIS Lumina imaging system at scheduled time points. To investigate the in vivo detection limit of MRSA infections, the NIRF images of mice (n = 3) infected by different concentrations of MRSA bacteria (105, 106, and 107 CFU) were obtained and assessed. Ex Vivo Biodistribution. A 500 μL amount of SiO2-Cy-Van nanoprobe PBS solution (163.64 mg/mL loaded with 150 μg of Cy and 2.05 μg of Van) was injected into MRSA-infected mice (n = 3) at 16 h postinfection, and various tissues including heart, liver, spleen, lung, kidney, and infected skin were extracted from the mice at 6 h postinjection. Finally, these tissues were imaged using an IVIS Lumina imaging system, and the NIRF signals from these tissues were measured to describe the ex vivo biodistribution of the dissociated polyelectrolyte layers. In Vivo PTT of MRSA Infections. To evaluate photothermal efficacy of our nanoprobe, 500 μL of SiO2-Cy-Van nanoprobe PBS solution (163.64 mg/mL loaded with 150 μg of Cy and 2.05 μg of Van) was injected into MRSA-infected mice (n = 3) at 16 h postinfection and irradiated by a NIR laser (808 nm, 1.5 W/cm2) for 5 min at 4 h postinjection. The festered areas of infection were recorded every 2 days. After 12 days of observation, the mice were executed and the tissues at the infected sites were dissected from the mice, fixed in a 4% formaldehyde solution for 24 h at room temperature, and frozen. Finally, the tissue slices were made, and H&E staining was subsequently conducted.

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obtained and the Avogadro constant, the average numbers of Cy and Van molecules immobilized on a SiO2-Cy-Van nanoprobe can be approximatively determined to be 618.15 and 3.97, respectively. In Vitro Biocompatibility Assay. The biocompatibility assay was carried out with the mouse embryonic fibroblast cells (NIH-3T3) (ATCC) by the MTT assay. Briefly, cells were seeded on the 96-well microplate at a density of 8000 cells/well and grew overnight, followed by treatment with SiO2-Cy-Van nanoprobes with different concentrations of Cy (1.25, 2.5, 5, 10, 20 μg/mL), respectively. After 24 h of incubation, the cell viability was evaluated with a microplate reader (Synergy NEO, BioTek). Photothermal Effects of Nanoprobes. To assess the photothermal effect of our nanoprobe, 1 mL of SiO2-Cy-Van water solutions with different concentrations of Cy (1, 2, 5, 10 μg/mL) was irradiated using a NIR laser (808 nm, 1.5 W/cm2) for 5 min, during which the sample temperature was continually monitored by a thermal imager, and about 1500 temperature data at different irradiation times were obtained in 300 s of irradiation. By using a line to connect these data points in the Origin software, photothermal heating curves of SiO2-CyVan nanoprobes could be plotted. Bacteria Culture. Escherichia coli (E. coli) (ATCC 8739), methicillin-resistant Staphylococcus aureus (MRSA) (ATCC 33591), and methicillin-sensitive Staphylococcus aureus (MSSA) (ATCC 6538) were employed in our experiments. The bacteria were cultured in Luria−Bertani broth medium (LB, Sigma-Aldrich) and harvested at the exponential growth phase prior to experiments. The concentration of bacteria was monitored by measuring the optical density (OD) at a wavelength of 600 nm. Prior to microbe-activated fluorescence and animal infection experiments, the OD600 values of bacteria PBS solutions were readjusted to 0.1, which corresponded to the concentrations of 4 × 108, 2 × 108, and 2 × 108 CFU/mL for E. coli, MRSA, and MSSA, respectively, obtained based on the colonycounting method. In Vitro Bacteria-Activated NIRF Imaging and Photothermal Antimicrobial Experiments. A 60.7 μL amount of SiO2-Cy or SiO2Cy-Van PBS solution with 494 μg/mL of Cy and 940 μL of bacteria suspension (E. coli or MRSA) with a concentration of 107 CFU/mL was mixed and incubated in an EP tube for 2 h. Then, 1 mL of bacteria suspension (107 CFU/mL) and 1 mL of fresh LB broth were then added and incubated for another 2 h. For bacteria-activated fluorescence experiments, the NIRF imaging of the mixture was performed using an IVIS Lumina imaging system. For photothermal antimicrobial experiments, the mixture was centrifuged at 3000 rpm for 10 min, and the bacteria precipitation was washed and finally dispersed in PBS. The bacteria suspension was subsequently irradiated using a NIR laser (808 nm, 1.5 W/cm2) for 5 min, and the antimicrobial performance was assessed by the live/dead bacterial staining assay and SEM-bacterial morphology study. Live/Dead Bacterial Staining Assay. The bacteria suspensions incubated with SiO2-Cy-Van nanoprobes before and after NIR laser irradiation were mixed with bacterial live/dead dye solution (0.5 mL, 1 μM of SYTO 9 and 5 μM of propidium iodide) (Invitrogen) for 20 min at room temperature and then imaged by confocal microscopy (TCS SP5, Leica). The SYTO 9 dye (green color) entered both intact and cell structure (cell wall and membrane)-damaged bacteria, while

ASSOCIATED CONTENT S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.7b00041. Computer simulation and additional experimental data (PDF)

AUTHOR INFORMATION Corresponding Authors

*E-mail: wfl[email protected] (W. Li). *E-mail: [email protected] (Y.-Q. Li). ORCID

Weifeng Li: 0000-0002-0244-2908 Yong-Qiang Li: 0000-0003-1551-3020 Chunying Chen: 0000-0002-6027-0315 4436

DOI: 10.1021/acsnano.7b00041 ACS Nano 2017, 11, 4428−4438

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ACS Nano Notes

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