Chemotaxis-Instructed Intracellular Staphylococcus aureus Infection

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Chemotaxis-Instructed Intracellular S. aureus Infection Detection by a Targeting and Self-Assembly Signal Enhanced Photoacoustic Probe Qian Cai, yue fei, Li-Ming Hu, Zhangjian Huang, Li-Li Li, and Hao Wang Nano Lett., Just Accepted Manuscript • Publication Date (Web): 28 Aug 2018 Downloaded from http://pubs.acs.org on August 29, 2018

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Chemotaxis-Instructed Intracellular S. aureus Infection Detection by a Targeting and SelfAssembly Signal-Enhanced Photoacoustic Probe Qian Cai†, ‡, ¶, Yue Fei†, §, ¶, Liming Hu ‡, Zhangjian Huang §, Li-Li Li †,* and Hao Wang †,* †

CAS Center for Excellence in Nanoscience, CAS Key Laboratory for Biomedical Effects of

Nanomaterials and Nanosafety, National Center for Nanoscience and Technology (NCNST),No. 11 Beiyitiao, Zhongguancun, Beijing, 100190, China ‡

College of Life Science and Bioengineering, Beijing University of Technology, Beijing 100124,

Beijing, China §

State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Drug Discovery for

Metabolic Diseases, China Pharmaceutical University, Nanjing 210009, China. KEYWORDS. Chlorophyll, self-assembly, photoacoustic, bacteria, bioimaging

ABSTRACT. Intracellular invasion and the survival of S. aureus in phagocytic cells has been regarded as one of the mechanisms that leads to the treatment failure of S. aureus infection and potential antibiotic resistance. The detection of infected phagocytic cells plays an important role in guiding antibiotic treatment and in reducing drug resistance. The development of a sensitive and specific imaging probe to visualize the intracellular bacteria is quite challenging. In this

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work, we report a photoacoustic agent (MPC) that is able to detect intracellular S. aureus infection through a dynamic process, including i) active targeting and internalization into macrophage cells, ii) specific molecular tailoring by caspase-1 in infected macrophage cells and iii) enhancement of the photoacoustic (PA) signal owing to molecular self-assembly. The PA signal per area of the ‘stimuli-induced assembly’ agent (MPC) increases more than 2-fold over that of the active targeting control agent (MPSC). Finally, based on this approach, the average PA signal in the infected site is enhanced by approximately 2.6-fold over that of the control site. We envision that this PA contrast agent may provide a new approach for the selective and sensitive diagnosis of an intracellular bacterial infection. Living systems can be regarded as the most sophisticated supramolecular organizations; for example, the DNA double helix, protein complexes, and fibrous assembled actin1-3. Mimicking the dynamic-assembly in vivo with well-ordered, biofunctionally endowed nanostructures is an unswerving goal of supramolecular chemistry.4-6 Inspired by natural chlorophyll molecules,7, 8 which modulate assembly/disassembly in light-harvesting, the artificially modified chlorophyll derivative displays a similar ordered molecular-level self-assembly in complex conditions in vivo.9-11 In our previous work, we demonstrated that the typical J-type self-assembly of chlorophyll derivatives induced a redshift in the absorption spectrum compared to that in the monomeric state, thus exhibiting an increased photoacoustic signal and improved signal-to-noise ratio.12 Furthermore, the good biocompatibility and biodegradation properties of chlorophyll derivatives offer promising potential in bioimaging.13-16 The innate immune system has evolved as a quick response mechanism specifically for pathogen-associated infection to protect the host from bacterial or viral infection.17 The chemotaxis of macrophages is a complex behavioral response to a chemoattractant at the


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bacterial infection site.18 According to this response, the macrophages are specifically directed to move to the infectious tissues from blood circulation. The classically activated macrophage (M1) will kill bacteria. However, typical long-term chronic inflammation induced by bacteria, such as S. aureus, enables survival inside the macrophage to protect themselves from being killed by antibiotics19. The diagnosis and detection of bacterial infection is hindered inside the macrophage, which leads it to escape immune eliminations, which is a great help for reducing antibiotic abuse and potential resistance.20-23 Recently, researchers have developed cell-mediated drug delivery carriers to reduce systemic toxicity, prolong blood circulation and facilitate migration across impermeable physiological barriers.24 However, based on the intrinsic chemotaxis property of macrophages, the rational design of probes for bacterial infection diagnosis is an important direction but is still only a concept. Here, we fabricate a chlorophyll-peptide-based photoacoustic agent (MPC) for in vivo bioimaging of bacterial infections (Figure 1). We endow MPC with a targeting ligand for specifically recognizing macrophages. Subsequently, the macrophage uptakes MPC and delivers it to the infectious site in a highly efficient manner. Upon the infection of macrophages by bacteria cells, the enzyme (i.e., caspase-1) is immediately activated and efficiently cleaves our designed peptide substrate. The resultant residues simultaneously self-assemble into J-type aggregates and accumulate inside of the macrophage. The assembly/aggregation-inducedretention (AIR) effect12 significantly enhances MPC accumulation at the infectious site. Consequently, the infection-induced AIR effect remarkably increases the contrast when we set up the detection window for the wavelength of aggregates. Finally, both the targeting enhancement and the chlorophyll aggregate-induced photoacoustic signal augment the increase and contribute to specific and sensitive bacterial infection bioimaging, thus enabling the


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detection of intracellular bacterial infection. Based on the in vivo self-assembly technique, we have faith that the invasive diagnosis of hard elimination bacterial infection may benefit reasonable antibiotic use that will reduce drug resistance. The synthesis and characterization of the peptide-chlorophyll molecule were reported in our previous paper25. The chlorophyll-peptide molecule presents as sandwich-like upon coordination with Cu2+. In this work, we find that when active chlorophyll-peptide targets onto macrophages, these dimers can be tailored by pathogen-associated molecular patterns (PAMPs) that are activated by caspase-1 to induce them to form a J-type assembly. The J-type assembly of chlorophyll derivatives significantly enhances the photoacoustic signal9, 10. In this regard, we design four chlorophyll-peptide molecules, which are named MPC, MPSC, PRC and PC, respectively (Table 1). The MPC is composed of three motifs, i.e., the targeting motif, the tailoring motif and the PA signaling motif (Figure 1). The MPC molecule enables entry into macrophage cells through the active targeting mechanism. Then, the peptide linker of MPC is cleaved by caspase-1 and the residue self-assembly via π-π and hydrophobic interactions when the macrophage is infected by bacteria. MPSC is one of the control molecules that lacks the caspase-1 tailoring property. PC is another control molecule, which shows an always-assembled state inside the noninfected and infected macrophages. PRC is the caspase-1 cleaved residue of the MPC. The synthetic procedures and their MALDI-TOF mass spectra are presented in the Supporting Information and in Figure S1-S4. First, the self-assembly and photoacoustic property of molecule PRC in solutions are studied. First, the job plot curve is determined by UV/vis spectrometry, which indicates that the PRC molecule coordinate with Cu2+ with a molar ratio of 2:1 in DMSO (Figure S5). The selfassembly procedure of PRC is carried out in the DMSO/H2O mixture with variable mixing ratios.


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The UV-vis absorption spectra shows that the original Qy absorption band of the dimeric PRC at a 676 nm decreases, broadens and redshifts to 697 nm with an increase in the H2O content up to 99% (volume ratio), which suggests the formation of J-type aggregates12 (Figure 2A). Moreover, unlike the usual circular dichroism (CD) spectra of chlorophyll-derivatives, which exhibit a conservative S-shaped and large-split CD signal at the corresponding Qy band at the aggregated state, the PRC dimers show an intense negative band at 680 nm (Figure 2B). The typical CD spectra observed in the PRC dimers give a single, strongly positive cotton peak at 423 nm and a negative one at 680 nm, respectively, which are associated with the Soret absorption band (Bband) and Qy-band11. This pattern can be attributed to the fingerprints on the dimeric geometries.7, 8 Upon formation of aggregates, the weaker characteristic bands (+423 nm and -680 nm) imply the racemization of the dimer, owing to self-assembly via π-π stacking and hydrophobic interaction. The morphology of the PRC aggregates is represented as fibrous structures with diameters of approximately 3-4 nm after standing overnight in an aqueous solution (Figure 2C) or PBS buffer (Figure S6). The photoacoustic property is further investigated upon self-assembly. We have discussed the mechanism of assembly induced PA signal enhancement in our previous works.9, 12 Furthermore, inherent parameters such as volume thermal expansivity, sound speed and heat capacity, as well as local optical property, affect the PA signal, and the PA signal is also closely correlated to the optical absorption coefficient, molar concentration and molar extinction coefficient of the PA contrast agent at certain wavelengths.9 Thus, the PA intensity of PRC dimers and their aggregates at variable wavelengths are monitored to determine the optimal detection window for further experiments (Figure 3A). For a better analysis of the PA intensity, the ratiometric PA signals of assembly verses the dimer is utilized. The ratiometric PA signal curves of PRC from 710 nm/680


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nm to 740 nm/680 nm in various mixture solutions of DMSO/H2O were measured, and the results are shown in Figure S7. Although the variation tendency of aggregated PA intensity from 710 nm to 740 nm are similar, the maximum difference of the ratiometric PA signal between aggregate and dimer is obvious at 710 nm/680 nm. The ratiometric PA images of PRC dimers, assemblies (Figure 3B) and the assembly procedure of PRC in mixture solutions of DMSO/H2O (Figure S8) are obtained according to the PA intensity ratio of 710 nm/680 nm. Compared to the UV-vis absorption of the Qy band during PRC assembly, the ratiometric PA images provide a noninvasive detection method for determining the PRC assembled state in the further cells and mice models. The caspase-1 enzyme activated self-assembly enhanced photoacoustic signal is further evaluated. As an alert mechanism of the innate immune system, caspase-1 is activated in the pathogen-associated molecular pattern (PAMPs) pathway26,

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activated caspase-1 as the specific target for monitoring the intracellular infection and thereby realize the chemotaxis-instructed bacterial infection detection in vivo. To verify the in situ assembly and PA enhancement by caspase-1 specific tailoring of the PA contrast agent, the S. aureus intracellular-infected macrophage cell model and its lysate are designed according to the literature28-30 and are schematically illustrated in Figure 3C. RAW 264.7 cells were chosen and infected by S. aureus through coculturing for 1 h. After clearance of the extracellular S. aureus with a dose of 50 µg/mL gentamycin, the infected macrophage cell model is obtained. Afterward, the infected cells are lysed by the low-osmosis method in 1/4 PBS buffer to prepare the cell lysate with activated caspase-1. Then, the UV-vis spectra of MPC and MPSC molecules that had been treated with a PBS buffer and a cell lysate were examined in parallel. In comparison to the Qy band of the PRC dimer and assembly, the MPC molecules exhibit an


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obviously responsive cleavage and the simultaneous self-assembly property in the cell lysate with the activated caspase-1 (Figure 3D). However, the noncleavable control (MPSC) shows only a slight redshift with an incubation time up to 2 h in the presence of cell lysate. The distinct property of the two molecules with different peptide linkers reveals that the designed peptide sequence of YVHDC indeed can be specifically recognized by caspase-110. The cleavage between the amino acids of Asp (D) and Cys (C) induces the MPC residues assembly, which is quite similar to the synthesized PRC molecule. Furthermore, ratiometric PA images of MPC, MPSC and PC in a PBS buffer and a cell lysate are tested separately, and the results indicated that our molecular design of MPC enables stabilization in the dimeric state without activated caspase-1 and in situ assembly once caspase-1 is activated in the lysate (Figure 3E). To further evaluate the imaging specificity of MPC to the infected macrophage, we performed the time-dependent caspase-1 activity during intracellular infection (Figure 4A). Based on the caspase-1 standard assay, the caspase-1 activity curve was carried out with cell lysates. As noted, caspase-1 was sensitively activated and the activity remained within 1 h, and then its activity reduced almost 50% up to 2 h. In fact, when administered MPC to the infected macrophage cells, the assembled residues of MPC induced a redshifted Qy band that appeared at 2 h (Figure 4B), which probably was attributed to the delayed effect of the entry into cells and the caspase-1 trigger. Importantly, with optimization of the PA detection window at 710 nm (Figure S9), the time-dependent photoacoustic intensity and fluorescence intensity comparison also reflect the dynamic assembly induced PA enhancement and fluorescence quenching (Figure 4C). Notably, the significant difference of MPC to MPSC after incubating with infected macrophage over 2 h (Figure 4D) indeed demonstrates that the enhancement of the PA signal is well correlated with the caspase-1 tailored self-assembly of the MPC residues. At the same time, the Cu2+


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coordinated P18 from the residues exhibit a J-type assembly, which is driven by a cleavagereduced molecular steric hindrance and increased hydrophobicity. The morphologies of the assemblies inside cells are confirmed the hypothesis above (Figure S10). What deserves to be noted is that the intracellular infection bioimaging exhibits no significant cytotoxicity to macrophage (Figure S11) cells when the molecular concentration is up to 160 µM. Efforts towards of this detection method may provide a new approach for precision medicine. Bacterial infection detection in vivo is studied. The photoacoustic (PA) tomography significantly improved the detection depth compared to optical imaging technology, which enables the reconstructed 3D images to be obtained through tomographic images. By using photoacoustic tomography technology, the tissue penetration depth can reach up to 50 mm.31 All the hind leg muscle infection models were carried out by orthotopic injection of implants next to the thighbone, which has a depth of approximately 5 mm. To further test this chemotaxisinstructed S. aureus infection PA contrast agent, we built three infection models, i.e., the infected macrophage model, S. aureus infection after antibiotic elimination, and S. aureus infection (detail description seen in Supporting Information). First, in order to clarify whether the MPC molecules can reach the intracellular infection and realize molecule tailoring-induced assembly, we intramuscularly injected infected RAW 264.7 cells (107 cells per injection) on the right leg and injected RAW 264.7 cells with the same quantity on the left leg as a control (Figure 5A). The 3D reconstruction of the PA signal intensity distribution at 8 h revealed that the i.v. injection of MPC molecules was well assembled and merged with the intracellular infection (Figure 5B). Moreover, the time-dependent PA image comparison (710 nm) of MPC and MPSC in Figure 5C clearly verified that the positive signal retention of MPC up to 8 h post injection was attributed to the dynamic assembly of MPC residues tailored by activated caspase-1. Moreover, within 12-


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36 h, the PA signals gradually reduced, which may have been due to the degradation or disassembly of the residues through hydrolysis12. The average PA intensity per area of MPC at 8 h is over 2-fold more than that of MPSC due to the assembly induced retention and assembly induced PA enhancement. Next, to assess the chemotaxis of macrophages for the infection detection, we built a S. aureus muscular infection (108 cfu per injection for 12 h) and then eliminated the extracellular bacteria through antibiotic treatment (100 µg per injection of gentamycin for 12 h). There are distinct differences between the infection site and the control site (PBS) in the PA images, which identifies that innate chemotaxis macrophages display the role of “cell carrier” delivery of the contrast agents to the infection site and assembly for PA detection (Figure S12). After all, the S. aureus infection model eventually demonstrates the chemotaxisinstructed infection of PA detection (Figure 5D). The quantitative result of the infection site and the control site (Figure S13) indicated that in the early infection stage, the phagocytosis of the activated macrophages32, which causes chemotaxis to the infection, greatly accumulated our assembled MPC residues in the infection site (2.6-fold over that of the control site). The photomicrographs of the infected muscles stained by H&E (Figure 5E) indicated the accumulation of leukocytes associated with the inflammation of the S. aureus invasion. The possibility of utilizing the chemotaxis mechanism in the innate immune system presents a remarkable advantage in the enhancement of the targeting efficiency for infection theranostics. Moreover, as mentioned before, the PA imaging signal was activated by the caspase-1 tailored and self-assembled MPC, which showed no direct relationship with the quantity of S. aureus. As with the early alert of intracellular infection, the caspase-1 activated PA imaging approach may provide early-stage detection of intracellular infection for antibiotic therapy guiding.


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In conclusion, this study demonstrates a chemotaxis-instructed bacterial infection bioimaging detection approach. This photoacoustic contrast agent can specifically accumulate inside the caspase-1-activated macrophage with a dynamic process of active targeting, molecular tailoring and sequential self-assembly. According to the significant cascade processes, the PA imaging of the assembled MPC signal showed a higher specificity to intracellular-infected macrophages and eventually enhanced imaging selectivity than that of the active targeting control (MPSC). This method may provide a possibility for sensitive alert in the early stage of bacterial infection through PA detection in vivo.


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Figure 1. Schematic representation of macrophage chemotaxis-instructed S. aureus infection detection in vivo and the molecular component of the probe (MPC). The imaging agent of MPC had three motifs: i) mannose as a ligand for macrophage targeting; ii) peptide YVHDC as a responsive motif for cleavage by the activated caspase-1, and the tailoring site was between amino acid D and C (red dot line); iii) P18-AL derivative as a PA signal motif for assembly induced photoacoustic signal enhancement. MPC is actively targeted to the mannose receptor of the macrophage cell. Under the pathogen-associated molecular patterns (PAMPs), the activated caspase-1 inside the macrophage cells specifically tailors MPC to induce self-assembly and retention. These assemblies show significant enhancement of the photoacoustic signal, which can be used as a sensitive alert early in bacterial infection through PA detection in vivo.


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Table 1. The molecular sequences and correlated properties of responsive self-assembly

Properties Name

Sequence a

Inside

Inside

infected

Responsiveness b macrophage c

macrophage

Mannose-Tyr-Val-HisMPC

Dimer

+

Assembly

Dimer

-

Dimer

Assembly

-

Assembly

Assembly

+

Assembly

Asp-Cys-Lys-(Ala-P18)

Mannose-Arg-Arg-AlaMPSC Lys-(Ala-P18)

PRC

Cys-Lys-(Ala-P18)

Tyr-Val-His-Asp-CysPC Lys-(Ala-P18) a

The conservative recognition sequence to caspase-1 is Tyr-Val-His-Asp-Cys (YVHDC), and the cleavage site is between Asp (D) and Cys (C). b The responsiveness is specific to activated caspase-1. c The infected macrophages (RAW 264.7 cells) are obtained by S. aureus infected RAW 264.7 cells for 1 h.


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Figure 2. The gradual assembly procedure of molecule PRC in solution. The UV-vis spectra A) and CD spectra B) of assembly procedure of molecule PRC in the mixture solution (H2O/DMSO) with different volume ratios (from 0% to 100%). The molecule concentration is 5 ╳ 10-5 M. C) The transmission electron microscope (TEM) image of PRC fibrous assemblies in the aqueous solution (H2O/DMSO; 95/5; v/v).


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Figure 3. Ratiometric PA imaging in vitro. A) The PA intensity curves of PRC dimers and assemblies. The appropriate ratiometric PA detection window is approximately 710-740 nm. Data represent the mean ± S.D. (n = 3). B) The ratiometric PA images (710 nm/680 nm) of PRC dimer and assembly. PRC dimers and assemblies are obtained in the DMSO and H2O solutions, respectively. C) Schematic illustration of the S. aureus-infected macrophage cell model and lysate. i) RAW 264.7 cells (108) are cocultured with 109 cfu S. aureus for 1 h for infection. ii) The infected RAW 264.7 cells are obtained by 50 µg/mL gentamycin eliminating the extracellular S. aureus for 30 min and then replacing the culture medium. iii) The lysate is obtained by the low-osmosis method in 1/4 PBS. D) The UV-vis spectra of MPC (solid lines) and MPSC (dotted lines) in buffer and cell lysate compared to PRC dimer and assembly (dashed lines). E) The ratiometric PA images (710 nm/680 nm) of MPC, MPSC and PC in the PBS buffer and infected macrophage lysates.


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Figure 4. The caspase-1-dependent PA detection of intracellular infection. A) The caspase-1 activity standard assay of infected macrophage cells. The detection solution is the lysate of RAW 264.7 cells infected by S. aureus at different infected times. B) The UV-vis spectra of cell lysates after 15 min, 30 min, 60 min and 120 min of infection during the treatment of MPC (concentration of 10-4 M). C) Time-courses of the PA signal enhancement and fluorescence signal quenching in S. aureus infected with RAW 264.7 cells. Data represent the mean ± S.D. (n = 3). D) The enhancement of the PA signal in infected macrophage after treated with MPSC and MPC. The S. aureus-infected RAW 264.7 cells (1 h infection) were incubated with MPSC or MPC (10-4 M) for 2 h. Triple asterisk (***) denotes statistical significance; ***, p < 0.001; onesample t-Test analysis. Data represent the mean ± S.D. (n = 3).


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Figure 5. Chemotaxis-instructed S. aureus infection PA detection in vivo. A) Schematic illustration of the mice model (intramuscular injection of infected RAW 264.7 cells) and photoacoustic tomography (PAT) detection. The infected RAW 264.7 cells were obtained with the same procedure as before. The mice model was built after intramuscular injection of infected RAW 264.7 (107 cells per injection) for 12 h. B) PA signal intensity distribution of infected RAW 264.7 cells in vivo after MPC administration with a dose of 35 mg/kg though i.v. injection for 8 h. C) PA images of intracellular infection in vivo between 1-36 h after i.v. administration of MPC and MPSC (35 mg/kg), respectively. The PA intensities per area of MPC and MPSC were calculated based on the red dotted circle area. D) PA images of muscular infection. The right leg was infected after intramuscular injection of 108 cfu S. aureus cells for 12 h. E) Representative micrographs of the histology of the muscle sections (H&E staining) of the S. aureus-infected and the control (PBS) groups. Black arrows indicate the leukocytes during inflammation of the S. aureus invasion. The number of mice in each group is 3.


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ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: XXXX. Additional details on materials, methods and synthesis schematics. Figures including MALDI-TOF, Job plot, TEM images, PA signals, cytotoxicity, additional in vivo PA images and statistical analysis. (PDF) AUTHOR INFORMATION Corresponding Author *Email: [email protected]. *Email: [email protected] ORCID Hao Wang: 0000-0002-1961-0787 Li-Li Li: 0000-0002-9793-3995 Author Contributions ¶

Q. Cai and Y. Fei contributed equally to this work. L.-L. Li and H. Wang conceived the project.

Q. Cai and Y. Fei performed experiments. L. Hu and Z. Huang discussed and provided valuable suggestions. The manuscript was written by L.-L. Li and H. Wang. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interests.


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ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (51725302, 31671028, 51573032, 21374026), the Science Fund for Creative Research Groups of the National Natural Science Foundation of China (11621505), the CAS Key Research Program for Frontier Sciences (QYZDJ-SSW-SLH022), the Key Project of Chinese Academy of Sciences in Cooperation with Foreign Enterprises (GJHZ1541) and the CAS Interdisciplinary Innovation Team. Prof. L.-L. L. thanks the Youth Innovation Promotion, CAS. ABBREVIATIONS MPC, Mannose-Tyr-Val-His-Asp-Cys-Lys-(Ala-P18) coordinated with Cu2+; MPSC, MannoseArg-Arg-Ala-Lys-(Ala-P18) coordinated with Cu2+; PRC, Cys-Lys-(Ala-P18) coordinated with Cu2+; PC, Tyr-Val-His-Asp-Cys-Lys-(Ala-P18). REFERENCES 1.

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BRIEFS Intracellular localization and the eventual infection bioimaging are essential and required for the guidance of antibiotic therapy and the reduction of drug-resistance. However, active targeting of phagocytic cells cannot specifically differentiate the activated cells, which are directly correlated with infection. Thus, we described a photoacoustic contrast agent (MPC) that is able to produce bioimaging of the intracellular infection through a dynamic process that is specific to the activated macrophage cells. We envision that our photoacoustic contrast agent and bioimaging method may provide a high selectivity and sensitive approach for the early diagnosis of bacterial infection. SYNOPSIS


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Chemotaxis-Instructed Intracellular Staphylococcus aureus Infection

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