Kill the Real with the Fake: Eliminate Intracellular Staphylococcus

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Kill the Real with the Fake: Eliminate Intracellular Staphylococcus aureus Using Nanoparticle Coated with Its Extracellular Vesicle Membrane as Active-Targeting Drug Carrier Feng Gao, Lulu Xu, Binqian Yang, Feng Fan, and Lihua Yang ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.8b00212 • Publication Date (Web): 29 Nov 2018 Downloaded from http://pubs.acs.org on December 1, 2018

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ACS Infectious Diseases

Kill the Real with the Fake: Eliminate Intracellular Staphylococcus aureus Using Nanoparticle Coated with Its Extracellular Vesicle Membrane as ActiveTargeting Drug Carrier Feng Gao, Lulu Xu, Binqian Yang, Feng Fan, and Lihua Yang* CAS Key Laboratory of Soft Matter Chemistry, School of Chemistry and Materials Science, University of Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui 230026 China * Corresponding authors: (L. Y.) [email protected]

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Staphylococcus aureus bacteremia is one of the most serious bacterial infection worldwide. Most complications of S. aureus bacteremia arise because the pathogen can survive inside host phagocytes especially macrophages, which makes elimination of intracellular S. aureus key to clinical success. Unfortunately, most antibiotics have poor cellular penetration capacity, which necessitates intracellular delivery of antibiotics.

We herein use nanoparticle coated with

membrane of extracellular vesicle secreted by S. aureus (i.e., NP@EV) as active-targeting antibiotic carrier, with counterparts coated with PEGylated lipid bilayer (i.e., NP@Lipo) or with membrane of outer membrane vesicle secreted by Escherichia coli (i.e., NP@OMV) included as controls. NP@EV is internalized at higher efficiency by S. aureus-infected macrophage than by naïve counterpart, whereas NP@Lipo and NP@OMV are not; instead, NP@OMV, but neither NP@EV nor NP@Lipo, is internalized at higher efficiency by E. coli-infected macrophage than by naïve counterpart. Moreover, when injected intravenously into mouse models, NP@EV, but neither NP@OMV nor NP@Lipo, exhibits significantly higher accumulations within four major organs (kidney, lung, spleen, and heart) bearing metastatic S. aureus infections than within healthy counterparts. These observations suggest that EV membrane coating of NP@EV endows the particle with active targeting capacity both in vitro and in vivo. As a result, when preloaded with antibiotics and intravenously administered to alleviate metastatic infection in S. aureus bacteremia-bearing mouse model, NP@EV confers its cargoes with strikingly improved efficacy; in doing so, NP@EV is significantly more efficient than both NP@Lipo and NP@OMV in kidney and lung ― which bear the highest metastatic bacterial burden and represent a most common sites for S. aureus infection, respectively. Such an active-targeting delivery platform may have implications in promoting clinical success on intracellular pathogen-associated complications.


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KEYWORDS: bacteria, drug delivery, nanoparticle, active targeting, extracellular vesicle


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Blood stream infection (i.e., bacteremia) caused by Staphylococcus aureus may be complicated by the most serious infections including endocarditis, osteomyelitis, necrotizing pneumonia, and sepsis

1-3,

which makes S. aureus bacteremia one of the most common and serious bacterial

infection worldwide4. Within minutes after the entry of S. aureus into bloodstream, the pathogen is taken up by host phagocytes ― primarily neutrophils and macrophages. While the majority of the bacteria are effectively killed by these cells, some survive inside the phagocytic cells, especially macrophages

4-9.

Such intracellular location not only protects S. aureus from killing

by antibiotics and by host defense mechanisms

1, 10-11

but also render the pathogen hijack the 4, 10.

Once

disseminated into tissues at distant sites including bones, joints, heart, kidneys, and lungs

1, 4, 10,

infected phagocytes as ‘Trojan horses’ for spreading infection via the bloodstream

intracellular S. aureus is associated with chronic or recurrent infections, including osteomyelitis 12,

recurrent rhinosinusitis 13, pulmonary infections 14 and endocarditis 15. Patients with reduced

counts of neutrophils are less likely to develop S. aureus bacteremia and disseminated diseases than those with normal counts of neutrophils

4, 16.

Clearly, to achieve clinical success on S.

aureus bacteremia, the elimination of intracellular S. aureus is crucial

10,

which requires

antibiotics to enter host cells. Unfortunately, most antibiotics have limited capacity of doing this 17.

It is thus imperative to develop intracellular delivery systems for antibiotics.

For this purpose, there are two major strategies in literature. One is to construct a responsively cleavable antibiotic-antibody conjugate

10.

This approach requires both a S. aureus-targeting

antibody and a responsively cleavable covalent bond for conjugating an antibiotic with the antibody. Nevertheless, the selection of pathogen-targeting antibody is effort consuming ― not to mention that the resulting antibody may bind with any Gram-positive bacteria rather than just S. aureus

10

― and the necessity of chemically conjugating the antibody with an antibiotic may


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limit the options on antibiotics. Due to an antibody’s inability to enter cells, the resulting antibody-antibiotic conjugate targets extracellular S. aureus; it opsonizes the extracellular S. aureus to promote its phagocytosis and, once within phagolysosome, releases its antibiotic responsively by phagolysosome proteases to kill the intracellular S. aureus

10.

Another major

strategy is to employ nanoparticles as antibiotic carriers and promote intracellular delivery by taking advantage of nanoparticles’ endocytosis

17.

Carrier composed of bacterial enzyme-

degradable polymer further enables targeted S. aureus eradication within infected macrophages without affecting the naïve macrophages

18.

These nanocarriers in general act by negative

targeting mechanism unless further functionalized with artificial targeting moieties, which inevitably involves extra efforts. Phagocytosis of a bacterium by a phagocyte leads to antigen presentation which enables rapid detection and clearance when the phagocyte is re-exposed to the same pathogen type.19-22 On the other hand, bacteria (both Gram-negative

23-24

and –positive

25)

naturally secret membrane

vesicles into the extracellular environment ― those secreted by Gram-positive bacteria are coined extracellular vesicles (Evs) while those by Gram-negtive bacteria are outer membrane vehicles (OMVs). These extracellular membrane vesicles contain a myriad of immunogenic antigens

26-27

and exhibit various pathogen associated-molecular patterns crucial for modulating

the host immune responses 28-29. Immunizing mice with EVs secreted by Gram-positive bacteria often elicit an immune response specific to EV components

30.

Recently, cloaking synthetic

nanoparticles with natural cellular membranes through a top-down fabrication approach represents one forefront of nanotechnology development.31-35 Coating gold nanoparticle with the membrane of OMVs secreted by Escherichia coli yields nanovaccine capable of generating strikingly enhanced immune responses compared to intact E. coli OMVs alone,34 suggesting that


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membrane-coating technique does not impact the immunomodulatory roles played by immunogenic antigens (e.g., proteins) naturally present in the membrane of E. coli OMVs. S. aureus EVs are enriched with membrane-associated proteins that play critical roles in bacteriahost interactions.25,

29-30

Having these in mind, we herein develop active targeting develivery

nanosystem by coating antibiotic-preloaded nanoparticle (NP-Antibiotic) with the membrane of EVs secreted by S. aureus and use the resulting NP-Antibiotic@EV particle as fake S. aureus for eliminating intracellular S. aureus (Figure 1). As a proof-of-concept, we coat a poly(lactic-co-glycolic acid) (PLGA) nanoparticle with the membrane of S. aureus EV and find that the resulting NP@EV particle actively targets both S. aureus infected macrophage in vitro and major organs bearing metastatic infections in S. aureus bacteremia-bearing mouse models. In contrast, neither counterpart coated with PEGylated lipid bilayer (i.e., NP@Lipo) nor that with the membrane of OMV secreted by E. coli (i.e., NP@OMV) demonstrate the observed active targeting capacity. As a result, when preloaded with antibiotics and administered intravenously to treat metastatic infection in S. aureus bacteremia-bearing mouse models, NP@EV confers its cargo with significantly improved efficacy in alleviating S. aureus burdens in major organs especially in kidney and lung, significantly more efficient in doing so than NP@Lipo and NP@OMV. RESULTS AND DISCUSSION Our NP@EV nanoparticle was prepared by fusing the membrane vesicle derived from S. aureus EV (i.e., EV ghost) over a PLGA nanoparticle (NP) with the membrane-coating technique

32, 35-36.

Though EV ghost has wide size distribution, the resulting NP@EV particle

exhibits relatively uniform size distribution as does the freshly-dispersed bare PLGA nanoparticle according to dynamic light scattering measurements (Figure 2a). The average


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hydrodynamic diameter of NP@EV (104.2 nm) is 11.2 nm larger than that of the bare PLGA nanoparticle (93.0 nm) (Figure 2a), and the extent of this diameter increase corresponds well to the thickness of two layers of an approximately 5.6 nm thick lipid membrane, indicative of successful coating of EV membrane over the PLGA nanoparticle. Consistently, zeta-potential measurements show that NP@EV has a zeta-potential (-24.7 mV) closer to that of EV ghost (22.0 mV), rather than that of bare PLGA NP (-34.2 mV) (Figure 2b). Unlike bare PLGA nanoparticle (Figure S1a), NP@EV exhibits a spherical core-shell structure under transmission electron microscopy, with a nanoparticle core being enclosed within a thin shell of approximately 5.3 nm in thickness (Figure 2c and Figure S4a-b), confirming successful membrane coating. The membrane coating was further verified with a protein bicinchoninic acid (BCA) assay; in contrast to bare PLGA nanoparticle which shows the absence of detectable protein content, NP@EV shows the presence of protein content as indicated by a significant increase in absorbance at 562 nm (Figure 2d), with a protein loading yield (i.e., the weight ratio of immobilized proteins on the PLGA nanoparticle) quantified to be approximately 7.0 ± 0.2 wt % (Figure 2d). SDS-PAGE gel electrophoresis, which examines protein contents, indicates that the membrane protein composition of S. aureus EV is mostly retained throughout the particle preparation procedure and can be identified on the resulting NP@EV particle (Figure 2e). Collectively, above results suggest that the as-proposed NP@EV particle has been successfully prepared, with membrane proteins of S. aureus EV being successful transferred to the nanoparticle’s surface. Owing to its EV membrane coating, NP@EV (but not bare PLGA nanoparticle (Figure S1b)) exhibits good colloidal stability in fetal bovine serum (FBS)supplemented phosphate buffer solution (PBS), as indicated by the negligible change in its hydrodynamic diameter over a span of 72 h (Figure 2f).


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To assess how membrane proteins in S. aureus EV affects NP@EV’s performance, we in the subsequent assays include two controls: They are NP@Lipo (counterpart coated with PEGylated lipid bilayer (Figure S2)) and NP@OMV (counterpart fused with membrane vesicle derived from E. coli OMV (Figure S3)). Characterizations confirm that NP@Lipo and NP@OMV have been successfully prepared with similar sizes, comparable zeta-potentials, similar spherical coreshell structures, and good colloidal stability (Figure S2-3 and Supporting Information) as does NP@EV (Figure 2). To assess whether NP@EV actively targets S. aureus infected-phagocytes in vitro, we use murine Ana-1 macrophage as the representative phagocyte and examine the in vitro cellular uptake efficiency both under fluorescence microscope and with fluorimeter. Our cellular uptake results show that NP@EV, but neither NP@Lipo nor NP@OMV, actively targets S. aureus infected-macrophage (Figure 3a-f and Figure S5a-g). Fluorescence microscopy images (Figure 3a) show that, after treatment with NP@EV (labeled with Dil, a red fluorescent probe), S. aureus infected macrophage cells (i.e., + S. aureus) exhibit apparently brighter red fluorescence than their naïve counterparts (i.e., uninfected), indicative of higher intracellular content of NP@EV. The observed difference in red fluorescence is further quantified with statistical analysis on fluorescence intensity within individual cells (Figure 3b): After treatment with Dil-labeled NP@EV, the average red fluorescence intensity per S. aureus infected macrophage cell is significantly higher than that per naïve counterpart, suggesting that S. aureus infection makes the macrophage cells uptake more NP@EV. In striking contrast, treatment with NP@Lipo (Dillabeled) fails in leading to the above difference in red fluorescence intensity (Figure 3a-b); instead, the red fluorescence intensity is similarly low in both S. aureus infected and naïve macrophages, indicating that NP@Lipo is internalized at low efficiency and in S. aureus


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infection irrelevant way. These results from the microscopy images and statistical analysis on individual cells in them are further confirmed with the quantitative cellular uptake assays using fluorimeter, which reveal the overall red fluorescence intensity of all inoculated macrophage cells (Figure 3e). Membrane proteins of S. aureus EVs play critical roles in bacteria-bacteria and bacteria-host interactions,25, 29-30 whereas a PEGylate lipid bilayer lacks any associated protein. Therefore, the ability of NP@EV to elicit the observed S. aureus infection promoted cellular uptake must arise because of its coating with S. aureus EV membrane. Similar as S. aureus EV membranes

25, 29-30,

E. coli OMV membranes contain diverse

immunogenic antigens critical for bacteria-host interactions

26-27.

One natural question to ask is

whether nanoparticle coated instead with E. coli OMV membrane elicits the observed S. aureus infection promoted cellular uptake. To exclude this possibility, we performed similar cellular uptake assays with NP@OMV but find that NP@OMV is internalized at similar efficiency by both S. aureus infected and naïve macrophages (Figure 3a-b and Figure 3e), indicating that the uptake of NP@OMV by macrophage is S. aureus infection irrelevant, as is the case with NP@Lipo. Still, the internalization of NP@OMV by macrophages (both S. aureus infected and naïve) is comparable to that of NP@EV by naïve macrophage and significantly higher than that of NP@Lipo (by both S. aureus infected and naïve macrophages) (Figure 3b), likely because both NP@OMV and NP@EV have coating materials of bacterial origin ― which naturally promotes uptake by phagocytes to certain extent. Intriguingly, the uptake of NP@OMV by macrophage is significantly affected by E. coli infection (Figure 3c-d and Figure 3f); after treatment with NP@OMV (Dil-labeled), E. coli infected macrophage (i.e., + E. coli) exhibits significantly brighter red fluorescence and higher red fluorescence intensity (averaged on per cell scale) than naïve macropage (i.e., uninfected). In


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stark contrast, E. coli infection fails in affecting the internalization of NP@EV and NP@Lipo (Figure 3c-d and Figure 3f). Indeed, the effects of E. coli infection on the internalization of NP@OMV verse that of NP@EV (Figure 3d and Figure 3f) mirrors those of S. aureus infection but in a converse way (Figure 3b and Figure 3d) and neither of them affects the internalization of NP@Lipo, which suggests that NP@EV actively targets S. aureus infected macrophage with high specificity (and same is true with NP@OMV toward E. coli infected counterpart when elimination of intracellular E. coli is necessary). We next examine whether NP@EV’s S. aureus infection promoted internalization necessarily provides its antibiotic cargo with improved efficacy in eliminating intracellular S. aureus. Vancomycin (Van) and rifampicin (Rif) are used as the model hydrophilic and hydrophobic antibiotics37, respectively, and are preloaded into the carrier’s polymeric matrix. Antibacterial assays show that the resulting NP-Van@EV acquires similar potency against intracellular S. aureus as free vancomycin (Figure 4a), despite of the former’s significantly weaker activity against planktonic S. aureus than the latter (Figure S8a). In fact, when being exposed to planktonic S. aureus, NP-Van@EV is the least potent among the examined four forumulations (in the order of decreasing potency, they are free vancomycin, NP-Van@OMV, NP-Van@Lipo, and NP-Van@EV) (Figure S8a); nevertheless, when treating intracellular S. aureus, NPVan@EV becomes comparably potent as the reast (Figure 4a). On the other hand, NP-Rif@EV is significantly more potent against intracellular S. aureus than all other three formulations (namely, free rifampicin, NP-Rif@OMV, and NP-Rif@Lipo) (Figure 4b), though it is just comparably potent against planktonic S. aureus as the rest (Figure S8b). Empty NP@EV lacks activity against intracellular S. aureus (Figure 4). Therefore, the observed activity of antibioticpreloaded NP@EV against intracellular S. aureus must arise because NP@EV actively delivers


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its cargo into the S. aureus infected macrophage (Figure 3) and therein releases its cargoes for antimicrobial efficacy (Figure S9). We next evaluate whether NP@EV actively targets tissues bearing metastatic infection in vivo. Specifically, we assess the biodistribution profiles of NP@EV (intravenously injected through the tail vein) in S. aureus bacteremia-bearing versus healthy mouse models (n = 6 per treatment group)

10, 32

and examine whether metastatic S. aureus infection within a major organ promotes

the particle’s accumulation within that organ (Figure 5). As controls, NP@OMV and NP@Lipo are included in similar assays. At 96-h after S. aureus inoculation (via intravenous injection through tail vein), metastatic infection has been successfully established in all five major organs because they unanimously have strikingly higher bacterial burdens than blood (Figure 5a) and, in the order of decreasing bacterial burden, they are kidney, liver, lung, spleen, and heart. Therefore, at 96-h after S. aureus inoculation, we assess the biodistribution profiles of each examined nanoparticle (Figure 5b).

For NP@EV (labeled with DiD), S. aureus infection

significantly promotes its accumulations within all examined organs (except liver) (Figure 5c), likely owing to the facilitation of S. aureus infection on internalization of NP@EV by macrophage (Figure 3) and the natural biodistribution profile of macrophage 38, suggesting active targeting capacity of NP@EV in vivo. To exclude the possibility that NP@EV’s active targeting capacity in vivo arises because phagocytes naturally seeks substances of bacterial origin, we perform similar assays but with NP@OMV and find that S. aureus infection fails in promoting NP@OMV’s accumulations within above organs (Figure 5e). (In fact, S. aureus infection even suppresses NP@OMV’s accumulation within spleen, with reasons we don’t know at the current stage.) Enhanced permeability and retention (EPR) effects are evident in tissues with infection 39-40.

To exclude the possibility that NP@EV’s active targeting capacity in vivo arises because of


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EPR effects within infected organs, we carry out similar assay but with NP@Lipo and observe undetectable effects of S. aureus infection on the accumulations of NP@Lipo within major organs (Figure 5d). Taken together, these results suggest that NP@EV actively targets S. aureus infected organs (except liver) owing to its coating with S. aureus EV membrane. We then evaluate whether antibiotic-preloaded NP@EV endows its antibiotic cargo with improved efficacy in alleviating metastatic infection in S. aureus bacteremia-bearing mouse models (n = 6 per treatment group).10, 32 Vancomycin (Van) and rifampicin (Rif) are used as the model antibiotics. The treatment was initiated at 24-h after S. aureus inoculation (Figure 6a) when the majority of the pathogen has acquired intracellular location as shelter

10

and repeated

twice to simulate a common practice in clinics (dosing antibiotics for three consecutive days before judging efficacy).

Antibiotic-preloaded NP@OMV and NP@Lipo are included as

controls in similar assays. Our results show that the resulting NP-Antibiotic@EV endows its cargoes with strikingly improved therapeutic efficacy in alleviating bacterial burdens in all five major organs (except when using NP-Van@EV for heart) (Figure 6). Although the other two nanoparticle formulations (NP-Antibiotic@OMV and NP-Antibiotic@Lipo) improves the efficacy of their cargoes to varying extent and depending on which organ under examination, NP-Antibiotic@EV is consistently the most efficient one within kidney (Figure 6), where S. aureus burden is the highest (Figure 5a). In kidney, NP-Van@EV reduces the bacterial burden by 1-2 orders-of-magnitude, whereas free vancomycin and NP-Van@Lipo are completely ineffective and NP-Van@OMV reduces the bacterial burden by 0.05, while *, ** and *** indicate p < 0.05, p < 0.01 and p < 0.005, respectively.


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Figure 6. Efficacy of antibiotic-preloaded nanoparticles in alleviating bacterial burdens in major organs (except liver) and blood of S. aureus bacteremia-bearing mouse models, with the performance of free antibiotics included for comparison. Each treatment group have 6 mice. (a) Schedule of S. aureus inoculation, three doses of drug formulations (administered intravenously), and sacrificing the mouse models for subsequent quantification on bacterial burdens (indicated by CFU counting). To ensure that the majority of the pathogen has acquired intracellular location as shelter, treatment was not initiated until 24 h after infection. (b) Vancomycin (Van) and (c) rifampicin (Rif) are used as model antibiotics, with doses kept constant at 10 mg/kg for vancomycin and at 0.5 mg/kg for rifampicin. Controls are those treated similarly but with sterile drug-free saline (i.e., (b) 5% glucose solution and (c) PBS), which indicate the bacterial inoculum sizes. The relative CFU (%) is defined as the ratio of the CFU within an organ of a drug-treated mouse model to that within the same organ type of 5%-glucose-treated counterpart. To better understand the efficacy of NP-Antibiotic@EV, the bacterial burdens within organs and blood of mouse models treated similarly but with free antibiotics, NP-Antibiotic@OMV, and NP-Antibiotic@Lipo are included for comparison. Data points are reported as mean ± standard deviation. The n.s. (not significant) represents p > 0.05. *, ** and *** indicate p < 0.05, p < 0.01 and p < 0.005, respectively.


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ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website at DOI: …... Supplementary figures: Figure S1-3, Characterizations on TEM image of (Figure S1) bare NP and S. aureus EV, (Figure S2) NP@Lipo, (Figure S3) NP@OMV, and (Figure S4) NP@EV and diverse dye-labeled nanoparticles; Figure S5, raw data for the fluorimeter-based cellular uptake assays; Figure S6, effects of endocytosis inhibitors on the nanoparticle internalization; Figure S7, In vitro cytotoxicity of empty nanoparticles; Figure S8, In vitro antibacterial assays against planktonic S. aureus; Figure S9, Drug release profiles; Figure S10, effects of NP@EV on the biodistribution profiles of S. aureus in mouse models; Figure S11-15, raw data on the therapeutic efficacy of (Figure S11-12) vancomycin-based and (Figure S13-15) rifampicin-based formulations in S. aureus bacteremia-bearing mouse models; Figure S16, blood biochemistry data; Figure S17, calibration curves and drug loading efficiencies.

Additional results and

discussion: Characterizations on NP@Lipo; Characterizations on NP@OMV; Effects of endocytosis inhibitors on the cellular uptake of nanoparticles; Antibiotic release profiles. Materials and methods: materials preparations and characterizations; cellular uptake assays; in vitro and in vivo antibacterial assays; biodistribution studies; in vitro cytotoxicity assays; blood biochemistry analysis; statistic analysis. References.

AUTHOR INFORMATION Corresponding Author


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* Lihua Yang: [email protected], +86-551-6360 6960. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest. Abbreviations EV, extracellular vesicle; OMV, outer membrane vesicle; Lipo, PEGylated liposome; NP, nanoparticle; PLGA, poly(lactic-co-glycolic acid); Van, vancomycin; Rif, rifampicin; CFU, colony

forming

units;

Tetramethylindodicarbocyanine

i.v.,

intravenous; Perchlorate;

DiD, Dil,

1,1'-Dioctadecyl-3,3,3',3'1,1'-Dioctadecyl-3,3,3',3'-

Tetramethylindocarbocyanine Perchlorate.

ACKNOWLEDGMENT We gratefully thank Professor Yucai Wang for use of his facilities. This work was supported in part by the National Natural Science Foundation of China (Grant 31671014, L.Y.), the Youth Innovation Promotion Association of Chinese Academy of Sciences (2014293, L.Y.), and the Ministry of Education of China (the Fundamental Research Funds for the Central Universities, WK3450000002).


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SYNOPSIS (Word Style “SN_Synopsis_TOC”).


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Kill the Real with the Fake: Eliminate Intracellular Staphylococcus

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