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Macrophage-Instructed Intracellular S. aureus Killing by Targeting Photodynamic Dimers Qian Cai, Yue Fei, Hong-Wei An, Xiao-Xiao Zhao, Yang Ma, Yong Cong, Liming Hu, Li-Li Li, and Hao Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b19056 • Publication Date (Web): 14 Feb 2018 Downloaded from http://pubs.acs.org on February 18, 2018

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ACS Applied Materials & Interfaces

Macrophage-Instructed

Intracellular

S.

aureus

Killing by Targeting Photodynamic Dimers †

‡

‡

‡

‡

‡

†

Qian Cai, Yue Fei, Hong-Wei An, Xiao-Xiao Zhao, Yang Ma, Yong Cong, Liming Hu,*, LiLi Li*,‡and Hao Wang*,‡ †

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

100124, China. ‡

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. KEYWORDS Peptide, Chlorophyll, Photodynamic therapy, Infection, Antibacterial agent

ABSTRACT The survival of S. aureus inside phagocytes is considered to be the sticking point of long-term chronic inflammation. Here, we fabricate peptide-chlorophyll based photodynamic therapy (PDT) agents with ‘sandwich’ dimeric structure to enhance PDT effect and active targeting property to eliminate intracellular infections, which could be seen as prospective antibacterial agents for inflammation.

Investigations in the early 50 years before have revealed that S. aureus is enable to invade and survive inside mammalian cells, primarily phagocytes and neutrophils, to preventing themselves from clearance.1, 2 Meanwhile, the incomplete clearance of intracellular S. aureus takes the host

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infected cells as ‘Trojan horses’ to disseminate the bacteria away from the initial site to the new ‘land of happiness’ for infection.3 This ‘Trojan horses’ phenomenon results in a significant enhancement of the antibiotic minimum inhibitory concentrations (MIC) for the intracellular bacteria causing the failure of the antibiotics in vitro and in vivo.1, 4, 5 Thus, the development of the antibacterial agents with intracellular activity against S. aureus poses challenges in the antimicrobial therapy. In the previous works, there are some strategies have proposed focusing on the intracellular activity during antimicrobial therapy, such as screening antimicrobial peptides with intracellular activity;6 delivering antibiotics into infected macrophages with nanoparticles;7, 8 and targeting S. aureus to follow and eliminate the bacteria inside infected macrophages by antibiotic.1 While, the realization and enhancement of intracellular activity for thorough clearance of the S. aureus infection are still under urgent requirements. The chlorophyll separated from leaves by organic solvent was introduced as pigment since 1818.9 A century ago, people have found the photosensitive property of the chlorophyll. Until recently, there are nearly 100 chlorophyll known today, which mainly obtained from anoxygenic bacteria, particularly from green bacteria.10 The excellent biocompatibility and outstanding photodynamic therapy (PDT) property of chlorophyll have been considered as promising therapeutic approach for cancer or infection therapy with ignorable microtrauma.11-17 This famous family of photosensitizers: chlorophyll-based molecule is well-known with high singlet oxygen quantum yield due to their large π-conjugated aromatic domains.13, 18 However, these molecules are easy to aggregate through π-π interactions in aqueous solution inducing the selfquenching effect, which reduced the 1O2 generation ability.19,

20

To address this issue, the

enhancement of the stability and singlet oxygen generation efficiency of the chlorophylls in


2

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aqueous medium is urgent and important. Beside improvement of traditional molecular structure, the rational design of the chlorophyll or porphyrin dimers21, 22 or assembles23, 24 to control the intracellular rotation contributing to their singlet oxygen production capability now is considered as a promising approach. Scheme 1. The chemical structure of MPepP18(Cu2+) dimer.

In

this

work,

we

developed

a

peptide-chlorophyll

based

photodynamic

dimer

(MPepP18(Cu2+)), which enable active targeting to macrophages, and then inducing receptor mediated endocytosis. The high stability and photodynamic effect efficiently eliminate S. aureus intracellularly. The molecular structure of the dimer was showed in Scheme 1. The purpurin 18 (P18) is coordinated with Cu2+ to form a ‘sandwich’ dimeric assembly with molecular ratio of 2:1, which identified by Job plot in Figure 1A. During formation of the dimeric equilibrium complex, the Q bands blue shifted arising from interference of π→π* transitions of the four frontier orbitals25 by the central Cu2+ (Figure S1). Fluorescence of the complex seemed


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dependent on the electronic orbital of the central metals, which could be explained the strongly quenched fluorescence by Cu2+ than Fe2+ in Figure 1B due to the heavy-atom effect.25 As known, the photosensitization of the chlorophyll and their coordinated complex to producing ROS was strongly dependent on the energy transfer capability to ground state oxygen, which could be enhanced by metal coordination. Besides, the conformational dynamics also influenced various photo physical processes, which subsequently controlled the quantum yield of singlet oxygen (Ф1O2) production.21,

26

Compared to the monomeric state of P18 and P18(Fe2+), the dimeric

conformation of P18(Cu2+) was indeed superior in singlet oxygen producing (Figure 1C). We inferred that the lower-energy conformer between P18 rings and Cu2+ was benefit for decrease of the excitation energy. The stronger binding constant 27, 28 of P18(Cu2+) than P18(Fe2+) (Figure S2) offered higher stability of P18 to Cu2+ .Then, efficient production of lived triplet state by dimers transferred to produce singlet oxygen.


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Figure 1. (A) Job plot to determine the mole fractions of P18 and Cu2+. Insert: Photos of equilibrium complexes in DMSO. (B) Fluorescence spectra of P18, equilibrium complexes of P18(Cu2+) and P18(Fe2+) in DMSO (10-5 M). (C) Plot of change in fluorescence of DPBF at Ex.420 nm vs irradiation time in presence of P18(Cu2+), P18(Fe2+) or P18 against MB (standard molecule) in DMSO. Chlorophyll-based molecules displayed readily assembled property in aqueous solution due to strong π-π and hydrophobic interactions. Both dimeric and monomeric state of the complex presented significant red-shifted and broadened of the Qy band associating with reduction of molar extinction coefficient (Figure 2A). While, the chirality transformation of dimers and monomers to assemblies were diametrically opposite (Figure 2B). CD spectra of the dimeric complex P18(Cu2+) exhibited chiral centers, which appeared Cotton effect peaks in B and Qy band region (425 nm and 676 nm). The assemblies of P18(Cu2+) strongly racemized to form achiral stacking structure. The monomeric P18(Fe2+) assembled into assembles displayed structural chirality showing bisignate signals in B and Q bands due to the excitonic coupling of the inherently chiral of monomers. Based on our previous works29, 30, the shift of Qy band predicted the π-π stacking assembly of chlorophyll. Thus, the peptide and mannose were used to modify molecules for enhancing stability and empowering targeting function. Comparing with P18(Cu2+) dimer in DMSO, the MPepP18(Cu2+) in aqueous buffer and cell lysate maintained the same Qy band position resulting from the stability of dimeric state in complex physiological medium (Figure S3).


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Figure 2. (A) Normalized UV-vis absorption spectra and CD spectra of the chirality of equilibrium complexes: P18(Cu2+) and P18(Fe2+) in DMSO and their assembles in aqueous solution (5% DMSO). (B) CD spectra of the chirality for P18(Cu2+)/P18(Fe2+) and their corresponding assembly.

Figure 3. (A) Time dependent shift of the Qy band for MPepP18(Cu2+) and MPepP18(Fe2+) in macrophage. Arrows: initial point-in-time of assembly. (B) Time dependent single oxygen


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attenuation in macrophage in dimeric and monomeric state. Statistic: 4 × 104 cells by flow cytometry. The solutions of MPepP18(Cu2+) and MPepP18(Fe2+) was PBS with 5% BSA added. As known, the assembly of chlorophyll-based molecules will significantly quench the singlet oxygen producing (Figure S4). Thus, stabilized monomeric distribution was the challenge task for maintaining long-term PDT property. Comparing the monomeric state MPepP18(Fe2+), the dimeric structure exhibited higher stabile ability inside macrophage. By monitoring the Qy band shifting in Figure 3A, the dimers of MPepP18(Cu2+) played longer stable time to 12 h, which was 6 folds longer than the stable time of 2 h for MPepP18(Fe2+). Meantime, dimers indeed showed enhanced singlet oxygen producing capability in macrophages in the original 15 min (Figure S5) counted by 4 × 104 cells in flow cytometry. Based on the stability, the single oxygen producing of dimers are less attenuated than monomers (Figure 3B), which was beneficial for high efficient clearance of intracellular infected bacteria. We speculated that maybe the higher binding constant of P18 to Cu2+ than to Fe2+ and the complex with BSA contribute to inhibition of aggregation, subsequently exhibited long-term ROS producing capability. Table 1. Extracellular and intracellular MICs of MPepP18(Cu2+) and vancomycin Extracellular MIC (µg/mL)a

a

Intracellular MIC (µg/mL)b

Van

MPepP18(Cu2+)c

Van

MPepP18(Cu2+)c

0.8 ± 0.2

6.2 ± 0.1

>100

25 ± 0.1

S. aureus in TSB media. b S. aureus inside RAW 264.7cells. c Irradiation for 5 min at 650 nm with 100 mW/cm2 laser.

To obtain the bacterial inhibition capability of the PDT agents and antibiotics, the macrophage extracellular and intracellular MICs were obtained respectively (Figure S6). As shown, the vancomycin performed excellent inhibition of S. aureus extracellularly, while it was inefficient at killing of S. aureus intracellularly. The similar conclusion was shown in majority of existing


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antibiotics, such as daptomycin, linezolid and rifampicin.1 Our designed PDT dimers MPepP18(Cu2+) had both efficient antibacterial effect for the intracellular and extracellular bacterial infection (Table 1). Depending on the specificity of targeting ligand, the PDT dimers can deliver a sufficient concentration into cells for bacterial killing (Figures S7-S9), while this concentration showed less cytotoxicity to macrophage (Figure S10). In addition, the receptormediated endocytosis pathway of the dimers (Figure 4) exhibited well co-localization to the phagocytic S. aureus, which might be attributed to the lysosome-phagosome fusion. Thus, the ROS producing by PDT dimers enable to efficiently kill the intracellular S. aureus in effective range (Figure 5).

Figure 4. Intracellular pathway of MPepP18(Cu2+) entering into macrophage. Confocal images of RAW 264.7 cells were obtained by treating with PBS under 37 ℃ and 4 ℃, 5 mM βcyclodextrin (β-CD, inhibitor for caveolae-mediated endocytosis), 2 mM amiloride (Inhibitor for micropinocytosis) and 450 mM sucrose (Hypertonic solution for the clathrin inhibition assay), respectively.


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Figure 5. Confocal images of intracellular ROS producing by MPepP18(Cu2+) in S. aureus infected macrophage. Green: DCFH-DA (2',7'-dichlorodihydrofluorescein diacetate); Red: Rhodamine B; Blue: Hoechst; Yellow: co-localization of ROS and S. aureus. Bars: 10 µm. To confirm the antibacterial effect in mice, the muscle S. aureus infected mice model were used (Figure 6A). After i.v. injection of PDT dimers and vancomycin with a dose of 110 mg/kg respectively, the group of MPepP18(Cu2+) were further treated with laser 1 h twice in the second infected day. Then, there were no additional treatment at 3rd and 4th day. The quantitative calculation of the infection in tissues were obtained by sacrificing the mice each day. The results presented that comparing to vancomycin, our PDT dimers exhibited significant inhibition of S. aureus infection in vivo, which lasted 4 days with once injection. However, the vancomycin showed inhibition activity at the 2nd day and the S. aureus infection relapsed from 3rd to 4th day. The protection of S. aureus in macrophage during vancomycin clearance and spread after antibiotic threat disappeared, maybe the two most possible reasons. The excited results


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confirmed that our designed targeting PDT dimers had high antibacterial activity both in vitro and in vivo, which could be used for complete clearance of S. aureus in infected tissue. Meantime, the acute toxicity of the dimers with 5 folds injection dose than therapy administration were investigated accordingly. The good biocompatibility of the PDT dimers exhibited no significant toxicity to organs, including heart, liver, spleen, lung and kidney (Figure 6B).


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Figure 6. In vivo evaluation of antibacterial efficiency and biocompatibility of PDT dimers. (A) Antibacterial activity of MPepP18(Cu2+) compared to vancomycin in S. aureus infected mice model. One i.v. injection at 2nd day with dose of 110 mg/kg for MPepP18(Cu2+) and 110 mg/kg for vancomycin. Laser: 650 nm, 100 mW/cm2 for 1 h twice at 2nd day. Statistical analysis: twoway ANOVA, **, p

Macrophage-Instructed Intracellular ... - ACS Publications

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