Antimicrobial Peptide-Reduced Gold Nanoclusters with Charge

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Antimicrobial Peptide-Reduced Gold Nanoclusters with Charge-Reversal Moieties for Bacterial Targeting and Imaging Dicky Pranantyo, Peng Liu, wenbin zhong, En-Tang Kang, and Mary B Chan-Park Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.9b00392 • Publication Date (Web): 15 Jul 2019 Downloaded from pubs.acs.org on July 18, 2019

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Antimicrobial Peptide-Reduced Gold Nanoclusters with Charge-Reversal Moieties for Bacterial Targeting and Imaging Dicky Pranantyo1, Peng Liu1, Wenbin Zhong2, En-Tang Kang1*, Mary B. ChanPark2* 1

Department of Chemical & Biomolecular Engineering National University of Singapore 4 Engineering Drive 4, Kent Ridge Singapore 117585 2

Centre of Antimicrobial Bioengineering School of Chemical and Biomedical Engineering Nanyang Technological University Singapore 637459

* Corresponding Authors E-mail: [email protected] (E.T.K) [email protected] (M.B.C.P) * ORCID En-Tang Kang: 0000-0003-0599-7834 Mary B. Chan-Park: 0000-0003-3761-7517

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Abstract To combat the increasing risk of infection by pathogenic bacteria, the new generation of antimicrobial agents are expected to exhibit non-metabolic killing mechanisms, high potency and biocompatibility. In this work, cysteine-terminated antimicrobial peptide (AMP) was employed directly as a reducing ligand to synthesize AMP-coated gold nanoclusters (Au NCs), bypassing the use of other reagents which might interfere with the efficacy of the resulting NCs. In addition to the use of biocompatible Au core, the primary amines of AMP coating were functionalized with anionic citraconyl moieties to further reduce cytotoxicity. The citraconyl amides could auto-cleave to reexpose the cationic amines at low pH. As a result, the AMP-coated Au NCs with citraconyl protection were stable and cytocompatible under physiological condition as determined by fluorescamine, hemolytic, cytotoxicity, and in vivo toxicology studies, but would switch into cationic bactericidal mode in an acidic environment commonly encountered at bacterial infection sites. Furthermore, the AMP-coated Au NCs system exhibited bacterial binding and photoluminescence features as determined by flow cytometry and confocal microscopy, which were useful for the detection and imaging of bacterial contamination. The AMP-coated Au NCs with citraconyl moieties therefore represent a ‘smart’ design of pH-responsive antimicrobial agents that can serve multiple functions of bacterial detection, bacterial imaging and anti-infection therapy.

Keywords: gold nanoclusters, photoluminescence, antimicrobial peptide, pHresponsive, theranostics.


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1. Introduction Diseases associated with microbial infections constantly become a major global healthcare problem. In addition to the use of conventional antibiotics to treat these infections, there is also a motivation to develop versatile antimicrobial agents that possess low risk to induce resistance.1 As a key constituent of the innate immune system in living organisms which offer immediate and effective defence against infections, antimicrobial peptides (AMPs) are alternative materials for the antimicrobial therapy.2 Frog-skin derivative esculentin-1a,3 human-skin derivative LL37,4 and hagfish epidermal-mucus derivative myxinidin5 exhibit strong antimicrobial activity towards a broad spectrum of Gram-positive and Gram-negative bacteria. Such naturally-extracted AMPs are usually present only in minute quantities and hence there is a great interest in the development of synthetic AMPs.6 The major components of AMPs are usually the cationic and hydrophobic segments. The cationic segment can destabilize the negatively-charged bacterial membrane, while the hydrophobic segment can induce peptide insertion into the bacterial lipid bilayer.7 Due to this killing mechanism via membrane disruption, AMPs typically lead to low frequency of emergence of resistant strains, unlike the commonly used antibiotics. However, the hydrophobic insertion function of synthetic AMPs often poses nonselective toxicity towards mammalian and other living cells.7 One approach to overcome this problem is by rational design for selective delivery of AMPs to the infection sites.

Nanomedicine is a rapidly growing technology which can provide numerous applications such as drug delivery,8,9 imaging,10 sensing,11,12 bio-purification,13 and tissue engineering.14,15 As such, the delivery of AMPs to the infection sites can be achieved by using either organic or inorganic nanocarriers. Poly(lactide-co-glycolide) 3 ACS Paragon Plus Environment


nanoparticles (NPs) was loaded with Esc(1-21) and Esc(1-21)-1c AMPs to treat the mouse lungs with cystic fibrosis infections.3 Porous silicon NPs were explored for systemic delivery of a proapoptotic AMP payload, while the NPs could undergo clearance through the liver, kidneys, and lungs of the tested animals.16 Most of these delivery systems required the AMPs to be loaded inside the cargo. As such, the bactericidal activities are dependent on the rate, extent, and mechanism of the controlled release. Another commonly used nanomaterials for in vitro and in vivo therapies are functionalized gold (Au) micro-/nanostructures, because they exhibit good biocompatibility with mammalian systems due to their high stability and chemical inertness.17 In term of molecular conformation, self-assembly of ultra-short lipopeptides on Au NPs exhibited a highly effective bactericidal tendency due to the protruding-oriented killing effectors brushes.18 The biocompatibility of these Au structures toward mammalian cells is retained even when the size of Au particles is reduced to a nanocluster (NC) scale.19 In addition, thiol-protected Au NCs that consist of several to a hundred atoms display luminescence, which is useful for imaging purpose.20,21 Moreover, at size below 2 nm, AuNCs were reported to exhibit antimicrobial activity through internalization that could lead to cell membrane destruction, DNA damage, and generation of intracellular reactive oxygen species (ROS).22-24 On the other hand, a recent study on the AuNCs coated with vancomycin antibiotic revealed that the controlled release of vancomycin was the predominant effect that contributed to the bactericidal activity.25 It has inspired to further explore the antimicrobial effects of AMP-functionalized Au NCs. The biocompatibility and luminescence properties of Au in the AMP-functionalized Au NCs not only are expected to reduce the cytotoxicity effects, but can also provide imaging function of the infection site.


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At the infection sites, the host immune system exhibits inflammatory responses to microorganisms by massive infiltration of neutrophils and macrophages. The effects of these responses and the bacterial metabolism result in localized acidosis, which could cause the pH of the infection sites to drop to as low as 5.5. This condition can be exploited to develop a pH-responsive drug delivery system, whereby the drug can remain stealthy during circulating in the body under physiological condition and resume its activity under the acidic condition of the local infection sites. In the design of antimicrobial agents involving highly cytotoxic AMPs or cationic polymers, this approach would be of particular benefit to selectively target bacteria. In antimicrobial studies, a library of amino peptides with β-carboxylic acid residue, such as histidine, aspartic acid and glutamic acid, exhibited pH-dependent protonation and net-charge changes, resulting in a major structure-function relationship.26 However, the design of supramolecules with intact amino moieties that can exclusively target bacteria remain a challenge due to the instantaneous nature of protonation and delicate differences between bacteria and cells membranes. For example, a histidine-rich designer peptide LAH4-L1 with pH-dependent net charge was useful to deliver a wide variety of cargo including nucleic acid, polypeptides, adeno-associated viruses, and nanodots via endosomal pathway, because it exhibited both strong antimicrobial and potent cellpenetrating activities.27 Another approach to regulate the net charge is by introducing temporary protection or deactivation of the protruding amino moieties. In a recent work, a charge-convertible polymer with zwitterionic groups that can switch to cationic quaternary ammonium groups under low pH was reported to target and kill bacteria with minimal toxicity towards mammalian blood and cells.28 These pHsensitive charge-conversion polymers have also been widely developed for drug delivery in cancer therapy.29



The aim of this work is to develop a pH-responsive antimicrobial agent that can remain stealthy and of low-toxicity under physiological (body) condition, but can kill bacteria on-demand at the infection sites. Firstly, cysteine-terminated AMP was employed as a ligand to reduce the Au(III) ions directly into AMP-coated Au NCs, avoiding the involvement of other molecules which may interfere with the antimicrobial efficacy. Composition is a crucial factor in the design of new antimicrobial agents, because the efficacy of an antimicrobial agent is often measured with a minute dosage. The cationic amines of AMP coating were then functionalized with anionic citraconyl moieties. The use of Au core and citraconyl protection reduce the hemo- and cytotoxicity of the AMP coating. The citraconyl amides are selfdegradable under acidic condition, resulting in the re-exposure of the cationic amines that can damage bacterial membranes upon contact. It was thus hypothesized that the cytraconyl-protected AMP-coated Au NCs could remain anionic and of low-toxicity under physiological condition, but would switch to cationic mode and kill bacteria at the acidic infection sites. The stability and cytocompatibility of the AMP-coated Au NCs with citraconyl protection towards mouse fibroblast cells was investigated. Upon cleaving of the citraconyl moieties, the antimicrobial activity of the AMP-coated NCs was challenged with Gram-negative and Gram-positive bacteria. The unique photoluminescence features of the AMP-coated Au NCs for bacteria detection and imaging were also presented.


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2. Experimental Section 2.1. Materials Gold (III) chloride trihydrate (HAuCl4·3H2O, 99.9%), citraconic anhydride (CA, 98%), fluorescamine, 2’,7’-dichlorofluorescein diacetate (DCFH-DA, 95%), 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

bromide

(MTT,

98%),

3,3’-

dipropylthiadicarbocyanine iodide (DiSC3(5), 98%), N-phenyl-1-naphthylamine (NPN, 98%), rhodamine B (95%), Triton X-100, sodium hydroxide (NaOH, 97%), hydrogen peroxide (H2O2, 3%-w in water), trifluoroacetic acid (TFA, 99%), chloroform (99.5%), and acetonitrile (99.9%) were purchased from Sigma-Aldrich Chem. Co. (St. Louis, MO). CysHHC10 (H-CKRWWKWIRW-NH2, 97%) peptide was purchased from China Peptides Co. Ltd. (Shanghai, China). 1,2-Distearoyl-snglycero-3-phosphocholine (DSPC, 99%) was purchased from Avanti Polar Lipids Inc. (Alabaster, AL). Hoechst 33342 and CyQUANT LDH cytotoxicity assay kit were purchased from Thermo Fisher Sci. Inc. (Waltham, MA). Spectra/Por regenerated cellulose dialysis membranes were purchased from Spectrum Chem. Co. (New Brunswick, NJ). Doubly-distilled water was produced from an Aquatron A4000D water distiller. Escherichia coli (E. coli, ATCC 25922), Pseudomonas aeruginosa (P. aeruginosa, ATCC 15692), Staphylococcus aureus (S. aureus, ATCC 25923), Staphylococcus epidermidis (S. epidermidis, ATCC 12228), and standard 3T3 mouse fibroblast cells were purchased from American Type Culture Collection (ATCC, Manassas, VA). Fresh whole blood of Wistar Hannover rat was purchased from InVivos Pte. Ltd. (Singapore).

2.2. Synthesis of the Peptide-reduced Gold Nanoclusters (Au-HHC NCs) CysHHC10 (31 mg, 0.02 mmol) was dissolved in 4.5 mL of doubly-distilled water. Aqueous solution of HAuCl4 (20 mM, 0.5 mL) was added dropwise under vigorous 7 ACS Paragon Plus Environment


stirring. The mixture was stirred at 700 rpm and heated at 70 °C for 24 h, during which the colour turned from fair red to pale yellow in less than 1 h. The mixture was then dialyzed against doubly-distilled water (MWCO 3.5 kDa) for 3 days to obtain aqueous solution of Au-HHC NCs, which can be stored at 4 °C for further use.

2.3. Synthesis of the Citraconyl-protected Au-HHC NCs (Au-HHC-CA NCs) The stock solution of Au-HHC (2.5 mL, 0.12 mmol of primary amine unit) was adjusted to pH 8.5 using 3 M NaOH solution. CA was gradually added dropwise to the Au-HHC solution under vigorous stirring. The reaction mixture was maintained at pH 8.5 using the 3 M NaOH solution and stirred overnight at room temperature. The mixture was then dialyzed against doubly-distilled water (pH 8.5, MWCO 3.5 kDa) for 3 days to obtain aqueous solution of Au-HHC-CA NCs, which can be stored at 4 °C for further use.

2.4. Characterization Chemical structures of the compounds in deuterated water were characterized by 1H NMR spectroscopy on a Bruker ARX 500 MHz spectrometer. Zeta potential of the compounds in aqueous solution at pH 8.5 and 22 °C was measured on a Malvern ZEN3600 zetasizer. Fluorescence analyses were performed on a Shimadzu RF-6000 spectrofluorophotometer. Transmission electron microscopy (TEM) images were obtained on a JEOL JEM-2100F field emission TEM. Elemental compositions of the lyophilized compounds were determined by X-ray photoelectron spectroscopy (XPS) on a Kratos AXIS Ultra DLD spectrometer, equipped with a monochromatized Al Kα X-ray source of 1486.71 eV photons with a constant dwell time of 100 ms, a pass energy of 40 eV, and a photoelectron take-off angle (α) of 90° with respect to the sample surface. 8 ACS Paragon Plus Environment

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2.5. Hydrolysis Study of Citraconic Amide of the Au-HHC-CA NCs The kinetics of citraconic amide hydrolysis was monitored using the fluorescamine protocol.30 Aqueous solution of Au-HHC-CA NCs (5 mg/mL, 50 µL) was added into 450 µL of citric acid-sodium phosphate buffer (pH 7.4). Similar procedure was carried out in pH 6.0, and pH 5.0 buffers. The solutions were agitated in an orbital shaker at 37 °C for 24 h. At each predetermined time interval, 10 µL aliquot was taken and diluted into 1 mL of borate buffer (0.1 M, pH 9.3) to quench the hydrolysis. This mixture was incubated with 10 µL of florescamine solution in N,Ndimethylformamide (2 mg/mL) at room temperature for 10 min. Its fluorescence intensity at 470 nm was measured under an excitation wavelength of 375 nm. Positive control (100% of exposed amine) was determined by applying this fluorescamine method on the solution after incubation of the Au-HHC-CA NCs in 0.01 M HCl overnight. Negative control (0% of exposed amine) was determined from the fluorescamine in blank buffer solution.

2.6. Antimicrobial Assays of the Au-HHC and Au-HHC-CA NCs Bacteria were cultured to a mid-log phase in the respective growth media at 37°C according to ATCC protocols, and diluted to 2 × 105 colony forming units (CFU)/mL in Mueller-Hinton broth (MHB). Stock solutions of sample compounds were incubated in PBS (pH 7.4 and 5.0) at a concentration of 512 μg/mL for 24 h. They were serially diluted by 2-fold in PBS, and 100 μL of each dilution was placed in a 96-well plate (Greiner Bio-one, Germany). Then, 100 μL of bacterial suspension was added to each compound solution. The plate was incubated at 37°C overnight and observed by naked eye. Bacterial growth made the suspension appear cloudy, while the suspension with no bacterial growth remained clear.31 The lowest concentration of the compound that inhibited the growth of bacteria was recorded as minimum 9 ACS Paragon Plus Environment


inhibitory concentration (MIC). The absorbance at 600-nm wavelength was measured as optical density on a Multiskan GO 1510 UV microplate spectrophotometer (Thermo Scientific, Waltham MA). Subsequently, 100 μL of the suspension in the wells with no visible growth was spread on a Mueller-Hinton agar and incubated at 37°C overnight to observe the viability of the bacteria. The lowest concentration of the compound that killed 99.9% of the initially inoculated bacteria was recorded as minimum bactericidal concentration (MBC).

2.7. Bacterial Membrane Perturbation and Liposome Leakage Assays The DiSC3(5) released assay32 and NPN uptake assay33 were performed as described previously with some modifications.34 Bacteria of mid-log growth-phase were resuspended at 2 × 106 CFU/mL in PBS containing 0.4 µM of DiSC3(5) or 10 µM of NPN. Then, 100 µL of bacterial suspension was added to each well of a black 96-well plate (Greiner Bio-One, Germany) and incubated at 37 °C for 1 h until the fluorescence level became stable. Subsequently, 100 µL of sample solution in PBS at various concentrations was added to each bacterial suspension and the plate was incubated at 37 °C for 1 h. The fluorescent intensities of DiSC3(5) (λex/λem at 610/710 nm) and NPN (λex/λem at 300/410 nm) in each well were measured on a Tecan Infinite M200 fluorescence microplate reader (Männedorf, Switzerland).

Liposome leakage assay was performed to study the membrane destabilization effect. Briefly, 10 mg of DSPC was dissolved in 0.5 mL of chloroform, slowly evaporated in a rotary evaporator, and dried under reduced pressure to form a thin lipid layer. The lipid layer was hydrated with 2 mL of doubly-distilled water containing 40 mM of rhodamine B, dispersed in an ultrasonic bath for 1 h, and dialyzed against doublydistilled water (MWCO 1 kDa) for 3 days. The lipid concentration was diluted to 5 10 ACS Paragon Plus Environment

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µM with PBS, and 100 µL of this liposome suspension was placed in each well of a black 96-well plate. Then, 100 µL of serially-diluted sample solutions in PBS was added to each liposome suspension. Blank PBS and 0.1%-v of Triton X-100 in PBS were added to the liposome suspension as negative and positive controls, respectively. The plate was incubated at 25 °C for 1 h and the fluorescence intensity was measured at λex/λem of 540/600 nm. The percentage of liposome damage was calculated as [(FS − FN) / (FP − FN)] × 100%, where FS, FN, and FP are the fluorescence intensity of the rhodamine B leakage from the incubated sample, negative control, and positive control, respectively.

2.8. Intracellular Reactive Oxygen Species (ROS) Generation Bacteria suspension (108 CFU/mL) was incubated with 0.1 mM of the sample compound in 1 mL of PBS at 37 °C for 2 h. DCFH-DA (10 µM, 1 µL) and Hoechst 33342 (1 µg/mL, 1 µL) were added to the suspension and agitated in an orbital shaker (200 rpm) at 37 °C for 15 min. The bacterial suspension was centrifuged at 10000 x g for 2 min, washed thrice with PBS, and resuspended in 1 mL of PBS. The intracellular non-specific esterase would cleave DCFH-DA into DCFH form, and in the presence of ROS, this DCFH would be further oxidized into the fluorescent 2’,7’dichlorofluorescein (DCF). Then 100 µL of the treated bacteria suspension was placed in a black 96-well plate, and the fluorescence intensity of the as-produced DCF was measured on a Tecan Infinite M200 microplate reader at an excitation/emission wavelength of 488/525 nm. The fluorescence intensity of this DCF would reflect the amount of intracellular ROS generated. The ROS amount was then normalized to the total number of bacteria cells, which was measured by the fluorescence intensity of Hoechst 33342 at an excitation/emission wavelength of 350/461 nm. The relative ROS production level was calculated by normalizing the ROS level of the sample11 ACS Paragon Plus Environment


treated bacteria with the production level of the blank control of bacteria (without sample treatment).

2.9. Assay of ROS-induced Degradation of AMP CysHHC10 peptide was dissolved in an aqueous solution of H2O2 (100 mM) at a concentration of 1 mg/mL. The degradation was allowed to proceed at 25 °C for 8 days. The solution was lyophilized after each predetermined time period. Buffer A (0.1%-v of TFA in doubly-distilled water) and buffer B (0.1%-v of TFA in acetonitrile) were prepared. The lyophilized compound was re-dissolved in buffer A at a concentration of 1 mg/mL and eluted through a 5 µm Agilent Eclipse Plus C18 column on an Agilent 1200 Infinity high-performance liquid chromatography (HPLC) using the mixed buffers at a linear gradient of 31%-50% (buffer B/A) in 15 min, with a flow rate of 1 mL/min, injection volume of 10 µL, and variable wavelength detector of 220 nm.

2.10. Bacterial Binding and Imaging by the Au-HHC and Au-HHC-CA NCs The sample compounds were incubated in PBS (pH 7.4 and 5.0) at a concentration of 50 μg/mL for 24 h. Bacterial cells were suspended in PBS to a concentration of 2 × 108 CFU/mL, and 0.5 mL of this bacterial suspension was added to 0.5 mL of the sample solutions. The mixture was agitated briefly using a vortex mixer and incubated at 37 °C for 3 h. The mixture was centrifuged at 2700 rpm for 10 min to remove the supernatant, washed and resuspended in 1 mL of PBS. The bacterial suspension was quantified using a CytoFLEX LX flow cytometer (Beckman Coulter, Indianapolis, IN) under 375-nm laser excitation with 525-nm fluorescence detector. To obtain qualitative images, one droplet of the bacterial suspension was spread on a glass slide, sandwiched with a cover slip, and observed under a Nikon Eclipse Ti-E confocal 12 ACS Paragon Plus Environment

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microscope (Tokyo, Japan). A Multi-Argon 408 laser was used as illumination source with 408 nm excitation wavelength and long-pass 417–477 nm emission filter settings for the blue signal.

2.11. Determination of Hemolytic Activity Sample compounds were serially diluted in 100 µL of PBS and placed in a 96-well plate. Then, 100 mL of fresh whole rat blood dispersion in PBS (8%-v) was added to each compound solution. The blood suspension was added to blank PBS as negative control, and to PBS containing 0.1%-v of Triton X-100 as positive control. The plate was incubated at 37 °C for 1 h. Each mixture was centrifuged at 1500 rpm for 10 min, and the optical absorbance of the supernatant was measured at 560-nm wavelength on a UV microplate spectrophotometer. The percentage of hemoglobin release was calculated as [(AS − AN) / (AP − AN)] × 100%, where AS, AN, and AP are the optical absorbance of the supernatant from the incubated sample, negative control, and positive control, respectively.

2.12. Cytotoxicity Assay Metabolic MTT assay was carried out to determine the cytotoxicity of the sample compounds. 3T3 fibroblast cells were cultured and resuspended in Dulbecco's modiﬁed Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 1 mM

L-glutamine,

and 100 IU/mL penicillin. Then, 100 µL of the suspension

containing 5000 cells were placed in each well of a 96-well plate. The plate was incubated in a humidified atmosphere of 95% air and 5% CO2 at 37°C for 24 h. Sample compounds were incubated in PBS (pH 7.4 and 5.0) at a concentration of 1024 μg/mL for 24 h and serially diluted by 2-fold in DMEM. The medium in each well containing the cells was replaced with 100 µL of the sample solutions, and the 13 ACS Paragon Plus Environment


plate was incubated at 37°C for 24 h. Nontoxic control experiment was carried out using the supplemented DMEM without any compound. The medium in each well was then replaced with 100 µL of MTT solution (0.5 mg/mL in supplemented DMEM). After additional incubation at 37°C for 4 h, the supernatant was aspirated and 100 µL of dimethyl sulfoxide was added to dissolve the internalized formazan crystal. After 15 min, the optical absorbance at 600-nm wavelength was measured using a UV microplate spectrophotometer and expressed as a percentage relative to the absorbance of the nontoxic control.

2.13. Lactate Dehydrogenase (LDH) Release Assay The CyQUANT LDH cytotoxicity assay kit contains lysis buffer, reaction mixture, and stop solution. Briefly, 100 µL of the 3T3 fibroblast suspension in DMEM containing 5000 cells were placed in each well of a 96-well plate and incubated in a humidified atmosphere of 95% air and 5% CO2 at 37°C for 24 h. In the maximum LDH activity wells, the medium was replaced with 100 µL of fresh DMEM. In the spontaneous LDH activity wells, the medium was replaced with a solution of 90 µL of fresh DMEM and 10 µL of sterile ultrapure water. In the compound-treated LDH activity wells, the medium was replaced with 100 µL of serially-diluted sample solutions in DMEM. The plate was incubated in a humidified atmosphere of 95% air and 5% CO2 at 37°C for 4 h. In the maximum LDH activity wells, 10 µL of the medium was aspirated out and 10 µL of the lysis buffer was added. The plate was further incubated in a humidified atmosphere of 95% air and 5% CO2 at 37°C for 1 h. Then, 50 µL of each medium was transferred to a new 96-well plate, and 50 µL of the reaction mixture was added to each well. The plate was incubated in the dark at 25 °C for 30 min, and 50 µL of the stop solution was added to each well. The absorbance was measured on a UV microplate spectrophotometer at 490 and 680 nm wavelength. 14 ACS Paragon Plus Environment

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The LDH activity was determined by subtracting the 680-nm absorbance (background) from the 490-nm absorbance. The percentage of LDH leakage was calculated as [(LDHC – LDHS) / (LDHM − LDHS)] × 100%, where LDHC, LDHS, and LDHM are the compound-treated, spontaneous, and maximum LDH activity, respectively.

2.14. In Vivo Toxicology Assays The CysHHC10, Au-HHC and Au-HHC-CA NPs in PBS solution were administered into female BALB/c mice (five mice per group) via intraperitoneal (i.p.) injection at a dosage of 5 mg/kg. Untreated mice were used as control. Mice weights were monitored daily until 7 days post-injection. After 7 days, mice blood was collected from submandibular vein for the blood biochemistry and complete blood panel analyses using Pointcare V2 blood chemistry analyzer and Celercare VH3 hematology analyzer (MNCHIP, China), respectively. Major organs (including spleen, liver, kidney, heart and lung) of the untreated and treated mice were harvested. After embedding, sectioning at 5 um thickness, and fixation, the tissues were stained with hematoxylin and eosin (H&E) stain and observed under digital microscope.

2.15. Statistical Analysis In general, the results were reported as mean ± standard deviation (SD) from three replicates, unless stated otherwise. The results were assessed statistically with Tukey post hoc test using one-way analysis of variance (ANOVA). Significant difference was accepted at p-value < 0.05.



3. Results and Discussion 3.1. Synthesis and Characterization of the Peptide-reduced and Citraconylprotected Gold Nanoclusters (Au-HHC and Au-HHC-CA NCs) Cysteine-terminated peptide, CysHHC10, was employed as a reducing-cumprotecting ligand to construct the Au-HHC NCs (Scheme 1). HHC10 is a highly active cationic short peptide which was optimally designed to combat a broad spectrum of superbugs,35 and has been successfully tested both in vitro and in vivo.36 Subsequent conversion of the cationic into anionic moieties was carried out by amidizing the primary amines on the corona of Au-HHC NCs with citraconic anhydride to obtain the Au-HHC-CA NCs. The 1H NMR spectrum of the Au-HHC NCs (Figure 1) resembles that of the CysHHC10 peptide with broadened lines, indicating that the peptide was embedded on the particles surface. In the Au-HHC-CA spectrum, the new chemical shifts characteristic of citraconyl amide at 2.0 and 5.5–5.7 ppm28 indicate successful amidation of the primary amines. The integrated areas of these new chemical shifts can be compared to that of the Au-HHC methyl proton shifts at 0.7 and 0.8 ppm to determine the degree of amidation. From the ratio of integrated peaks, it is estimated that each NC was decorated with 5–6 molecules of CA. During the reduction of Au(III) into Au(0) and simultaneous aggregation of the Au(I)-thiolate complexes on the Au(0) surface, the appearance of reaction mixture changed from transparent red to colourless within minutes, and then gradually turned pale yellow (Figure 2a). Similar phenomenon was observed previously during the synthesis of Au NCs using glutathione as a reducing agent.37 Upon subsequent amidation, the mixture became translucent white. In the XPS wide-scan spectra of the Au-HHC and Au-HHC CA NCs (Figure S1), the characteristic signals of Au 4f7/2 and Au 4f5/2 are discernible at the binding energies (BEs) of 84 and 88 eV, respectively,38 implying that the thicknesses of HHC and HHC-CA coatings on the Au NCs core are


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less than the XPS probing depth of ~8 nm.39,40 In the S 2p core-level spectrum of the CysHHC10 peptide, a spin-orbit split doublet (S 2p3/2 and S 2p1/2) with respective BEs at 163.2 and 164.3 eV and a spectral component area ratio of 2:1 is associated with the unbound sulfur species of the thiol terminal.39 Upon chelation with Au complexes, a new spin-orbit split doublet emerges in the S 2p core-level spectra of the Au-HHC and Au-HHC-CA NCs at the BEs of 162 and 163 eV, attributable to the sulfur species bound on the Au surface by chemisorption (S-Au).41,42 A smaller degree of weakly physisorbed thiols remain on the NCs surface, which presumably arise due to the high density of peptide molecules that formed a self-assembled monolayer on the surface.41 The XPS peak area ratio was used to determine elemental stoichiometry of the compounds.43 Upon amidation of the Au-HHC to Au-HHC-CA NCs, the [N]/[C] ratio decreased from 0.24 to 0.17 due to protection of the primary amines by the citraconyl moieties (Table S1).

Au NCs that consist of several to a hundred atoms exhibit fluorescence at various wavelengths.21 Upon excitation by near-UV of 365-nm wavelength, the Au-HHC and Au-HHC-CA NCs emit blue (426 nm) and yellow (456 nm) luminescence, respectively (Figure 2b,3a). An increase in the ligand length of NCs has resulted in the shift of photoemission to a higher wavelength. This result is in agreement with the previous observation that the emission peak of silver (Ag) NCs shifted to higher wavelength upon functionalization with daptomycin.44 From the transmission electron microscopy (TEM) images, the Au-HHC and Au-HHC-CA NCs exhibit well-defined size distributions with average diameters of about 2.4 and 2.6 nm, respectively (Figure 2c,d). At pH 7.4, the hydrodynamic sizes of the Au-HHC and Au-HHC-CA NCs increase to about 4.2 and 6.8 nm, respectively (Table 1), due to the hydration effect of



the coatings. The zeta potential (ζ) of Au-HHC NCs was recorded at 31.4 mV, slightly higher than that of the free peptide presumably due to the particles formation. Upon amidation, the ζ value of the Au-HHC-CA NCs dropped to -42.3 mV, implying thorough protection of the primary amine moieties with the charge-reversal CA molecules. The amount of Au metal and carbon residues of the peptide-functionalized NCs was determined by thermogravimetric analysis (TGA, Figure 3b), which has been commonly employed as a valid method to estimate the actual content of surface coatings on metal nanoparticles.45 The Au-HHC NCs contains about 83% mass fraction of peptide, whereas the Au-HHC-CA NCs contains about 89% mass fraction of peptide coating and CA moieties (Table 1).

In the presence of β-carboxylic acid terminal, amide bonds are acid labile and degradable at low pH.29,46 As such, the Au-HHC-CA NCs exhibited a charge-reversal conversion from anionic carboxylic acid to cationic primary amine at low pH. The extent of degradation of citraconic amides was studied by incubating the Au-HHC-CA NCs in buffers of different pH. The percentage of primary amine exposed as a result from the degradation was monitored using the amine-reactive fluorescamine dye. In a buffer solution of pH 5.0, up to 95% of the citraconic amides were hydrolysed to form primary amines within 24 h (Figure 3c). On the contrary, only 6% of the amides were hydrolysed after 24-h of incubation in the pH 7.4 buffer, demonstrating the pHdependent charge-reversal conversion of the Au-HHC-CA NCs. These observations are in agreement with the previous study on the degradation of β-carboxylic amides of the zwitterionic poly(N’-citraconyl-2-(3-aminopropyl-N,N-dimethylammonium)ethyl methacrylate).28 It is thus expected that the Au-HHC-CA NCs will remain stable and biocompatible by retaining their anionic charges under the physiological condition


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(pH 7.4). However, they will expose the primary amines which are readily protonated into cationic ammoniums in the low pH environment of the bacterial infection sites.

3.2. Antimicrobial activities and bacterial-imaging features of the Au-HHC and AuHHC-CA NCs Antimicrobial properties of the NCs towards various Gram-negative (Escherichia coli and Pseudomonas aeruginosa) and Gram-positive (Staphylococcus aureus and epidermidis) bacteria were studied using the standard method of dilution antimicrobial susceptibility.31 The native CysHHC10 peptide shows minimum inhibitory concentration (MIC) values of 8–64 µg/mL towards the bacteria challenged (Table 2), which is consistent with the reported results.47,48 In comparison to the free peptide, the Au-HHC NCs exhibited a higher antimicrobial activity towards the bacteria challenged, with MIC values at half (4–16 µg/mL) of that of the free peptide. The increase in antimicrobial efficacy could probably be attributed to: (1) the generation of reactive oxygen species (ROS) upon internalization of the Au-HHC NCs into the bacterial cells and (2) the more compact and protruding orientation of cationic moieties on the particles surface which provided greater membrane binding and destabilization. The Au-HHC NCs could incite 4.7- and 3.1-fold increase in intracellular ROS generation in E. coli and S. aureus, respectively, as compared to the unperturbed bacteria in the blank control (Figure 3d). These results are consistent with the previous study, in which mercaptohexanoic acid-reduced Au NCs were reported to penetrate bacterial membranes and induce metabolic imbalance by inflicting a 3-fold increase in ROS production within the bacterial cells.22 Meanwhile, the CysHHC10 peptide induced only 1.5-fold increase in ROS towards the bacteria, which is understandable because the main bactericidal activity of the cationic peptide is contributed by electrostatic disruption of the cell membrane and not by the ROS 19 ACS Paragon Plus Environment


production. Upon incubation of the CysHHC10 peptide in 100 mM of aqueous H2O2 for 8 days, the high-performance liquid chromatography (HPLC) elugrams show a peak split with comparable absorbance values (Figure S2). The second elution is associated with the disulfide formation of two peptide molecules, due to oxidation by the highly concentrated H2O2 as a strong ROS. Other than the peak split, there is no major difference in their HPLC elugrams, implying that ROS did not induce any significant degradation to the peptide.

Inner membrane depolarization was studied using 3,3’-dipropylthiadicarbocyanine iodide (DiSC3(5)) dye. DiSC3(5) accumulates in cells on hyperpolarized cytoplasmic membranes where it exhibits self-quenched fluorescence.32 When the membrane loses its potential or integrity, the dye is released from the cell and fluorescence is observed. Outer membrane permeabilization was investigated using N-phenyl-1naphthylamine (NPN), a small hydrophobic molecule that fluoresces strongly in the hydrophobic environment of lipid membrane, but only fluoresces weakly in aqueous environment.34 NPN naturally accumulates on the compromised lipid membranes, but it is excluded from the intact bacterial outer membranes.33 Upon bacterial incubation with the CysHHC10, the fluorescence intensities of DiSC3(5) and NPN showed proportional dependency on the peptide concentration, demonstrating its potency to compromise bacterial membrane (Figure 4b). In comparison to the CysHHC10 peptide, the Au-HHC NCs inflicted stronger cytoplasmic membrane depolarization and outer membrane permeabilization towards Gram-positive and Gram-negative bacteria, because clusters formation of the peptide gave rise to denser, more compact, and highly robust cationic charges with protruding orientation.


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Lipids are important constituents of bacterial membrane, in which Gram-positive bacteria have a single lipid bilayer, while Gram-negative bacteria have two concentric lipid bilayer membranes with different lipid compositions in each membrane.49 Interaction of the sample compounds with the microbes membrane was also investigated using rhodamine B leakage from liposome vesicles as a model of phospholipid bilayer. An increase in concentration of the antimicrobial compounds resulted in higher damage to liposome vesicles (Figure S3a), due to the presence of a larger amount of cationic charge to destabilize the liposome membranes. At the same concentration, the Au-HHC NCs induced larger liposomal damage than the CysHHC10 peptide, demonstrating that the higher density and protruding orientation of the amine moieties on the NCs surface have resulted in greater destabilization of the phospholipid membrane. In a recent study, daptomycin-conjugated Au NCs has been reported to exhibit higher antibacterial efficacy toward methicillin-resistant S. aureus, due to enhanced membrane disruption by the localized daptomycin within the structures and increased ROS generation within the bacteria.50 Au NPs functionalized with the extracted peptide from Vespa orientalis wasp venom have also been reported to have lower MIC values toward various bacteria, as compared to the free peptide without embedment on Au NPs.51 These results support the mechanistic perspective that particles or clusters formation of AMPs gives rise to higher antimicrobial activity.

The charge-reversal feature of the Au-HHC-CA NCs was investigated by pretreatment in PBS of different pH prior to the antibacterial assay. Upon pretreatment at pH 7.4, the Au-HHC-CA NCs did not exhibit apparent antimicrobial activity up to the highest concentration tested at 512 µg/mL (Figure 4a). However, with pretreatment at pH 5.0, the Au-HHC-CA NCs displayed MIC values comparable



to that of the free peptide, as a result of hydrolysis of the citraconic amides to regenerate the native primary amines. This result suggests that the Au-HHC-CA NCs can undergo the charge reversal without losing their antimicrobial potency. The antimicrobial effect of Au-HHC-CA NCs after hydrolysis treatment was lower than that of the Au-HHC NCs, implying that there was a small fraction of persistent cytraconyl moieties which were not fully cleaved after the treatment. According to the fluorescamine assay, treatment of Au-HHC-CA NCs in citrate buffer at pH 5.0 for 24 h could liberate 95% of primary amines as compared to the control treatment (Figure 3c). Moreover, in comparison to the Au-HHC NCs, the Au-HHC-CA NCs also inflicted lower intracellular ROS generation in E. coli and S. aureus (at 2.4- and 2.0fold increase, respectively) from the unperturbed bacteria controls (Figure 3d), resulting in a lower antimicrobial activity as well. The minimum bactericidal concentration (MBC, Table S2) values of the free peptide and peptide-reduced Au NCs were consistent with their respective MIC values, which further confirm their efficacy in eradicating the pathogens.

In addition to antimicrobial activity, the AMP-reduced Au NCs also possess bacterial binding and imaging features. Gram-negative E. coli and Gram-positive S. aureus were each incubated with the Au-HHC and Au-HHC-CA NCs and the fluorescence intensities of the bacteria were measured by flow cytometry. Each suspension is expected to exhibit a specific level of mean fluorescence intensity (MFI) based on the amount of NCs bound to the bacteria. From the flow cytometric histograms (Figure 5a,b), the bacterial suspension incubated with Au-HHC NCs showed the highest MFI level among all other compounds, indicating their abundance on the bacterial cells. Upon pretreatment in PBS of pH 7.4, the Au-HHC-CA NCs also showed some


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interaction with the bacteria, albeit the absence of their antimicrobial effect discussed earlier. However, after being pretreated in PBS of pH 5.0, their MFI level increased, suggesting that a larger amount of the NCs had bound to the bacteria due to exposure of the primary amines. Each suspension was then centrifuged at 2700 rpm to precipitate the bacteria. Under 365-nm irradiation, bacteria sediment appeared dull while the bacterial sediments with the NCs incubation exhibited the fluorescence effect (Figure 5c,d). It is noted that the NCs dispersion without bacteria was not precipitated upon centrifugation at 2700 rpm. Such strong bacterial binding and luminescence properties of the Au-HHC and Au-HHC-CA NCs (at pH 5.0) can potentially be developed for rapid detection and imaging of pathogens in water, biological fluids, and infected environment. To demonstrate the bacterial imaging feature of these NCs systems, a droplet of each bacterial suspension after being incubated with the NCs was examined under confocal microscope (Figure 6). Abundant bacterial cells were discernible in the sediment with Au-HHC NCs incubation, and only a smaller amount of bacteria were bound to the Au-HHC-CA NCs pretreated in PBS of pH 5.0. Only a few spots of bacterial cells could be observed after incubation with the Au-HHC-CA NCs pretreated in pH 7.4. These results imply a different level of binding capacity for each compounds, consistent with the data obtained from antimicrobial effects and flow cytometry.

3.3. Hemotoxicity and Cytotoxicity of the Au-HHC and Au-HHC-CA NCs Hemolytic activity of the compounds toward rat whole blood was determined as one of the toxicity parameters of antimicrobial agents. The CysHHC10 peptide showed a strong hemolytic activity (Figure 7a) and incited 50% lysis of the blood cells at a concentration of 670 µg/mL (HC50, Table 2). Upon incorporation on Au NCs, the HC50 value of the Au-HHC NCs increased to 765 µg/mL. The Au-HHC-CA NCs did 23 ACS Paragon Plus Environment


not show any hemolytic activity up to the highest concentration tested at 1024 µg/mL, indicating that the cytraconyl protection was effective to shield the amine moieties.

Cytotoxicity of the NCs towards mouse 3T3 fibroblast cells as the standard mammalian model was determined by measuring the half maximal inhibitory concentration (IC50) using the metabolic methylthiazolyldiphenyl-tetrazolium bromide (MTT) protocol.6 The CysHHC10 peptide recorded an IC50 value at 43 µg/mL (Table 2), consistent with the literature report.48 The Au-HHC NCs showed lower cytotoxicity than the free peptide, presumably due to the inclusion of biocompatible Au NCs (Figure 7b). After pretreatment in PBS of pH 5.0, the Au-HHC-CA NCs showed a higher IC50 value at 102 µg/mL as compared to that of the Au-HHC NCs at 56 µg/mL, indicating lower amount of surface cationic charges. However, after pretreatment in PBS of pH 7.4, the Au-HHC-CA NCs did not show any indication of cytotoxicity up to the highest concentration tested at 1024 µg/mL. These results indicate that the Au-HHC-CA NCs remain stable and retain their anionic protection characteristics under physiological condition. Selectivity value (IC50/MIC) is the ratio of cytocompatibility to bactericidal efficacy, and is commonly determined to express the therapeutic index of antimicrobial compounds. The CysHHC peptide exhibited poor selectivity of 1 to 5 towards various bacteria (Table 2). The formation of AuHHC and Au-HHC-CA NCs dramatically improved the selectivity by 2- to 4-fold over the free peptide. These results indicate that the incorporations of Au metal and charge-reversal moieties are a promising strategy to improve the selectivity of AMP. The true potential of Au-HHC-CA NCs lies on the on-demand charge conversion, whereby they remain cytocompatible under physiological pH and turn bactericidal in the low pH environment, such as at the bacterial infection sites.


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Lactate dehydrogenase (LDH) is a cytosolic enzyme present in many cell types, which is released upon damage to the plasma membrane.52 The extracellular LDH can be quantified by a coupled enzymatic reaction, in which LDH catalyses the conversion of lactate to pyruvate via NAD+ reduction to NADH. Oxidation of NADH by diaphorase leads to the reduction of a tetrazolium salt (INT) to a red formazan product that is measurable by spectrophotometry. The level of formazan formation is directly proportional to the amount of LDH release, which is indicative of cytotoxicity. In general, the Au-HHC NCs induced lower LDH leakage from 3T3 fibroblasts as compared to the free CysHHC10 peptide (Figure S3b), presumably due to the mass incorporation of the more cytocompatible Au NCs. On the other hand, the Au-HHCCA NCs did not show any significant LDH leakage up to the highest concentration tested at 1024 µg/mL. These results indicated that the cytraconyl protecting group was effective in reducing cell membrane perturbation by the amino moieties of the peptide.

3.4. In Vivo Toxicology Study of the Au-HHC and Au-HHC-CA NCs In vivo toxicity of the CysHHC10, Au-HHC, and Au-HHC-CA NCs towards BALB/c mice were evaluated after a single-dose intraperitoneal (i.p.) administration through the tail vein. All mice showed normal behaviour, and no mouse died or became moribund after 7 days post-injection. All mice did not show any obvious weight loss up to 7 days of examination (Figure S4). From the blood biochemistry analysis, no nephro- or hepato-toxicity was induced by the sample compounds, as demonstrated by the negligible variation of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and blood urea nitrogen (BUN) level for all the three treated groups (Figure S5). The routine hematology analysis showed no significant change in blood panel 25 ACS Paragon Plus Environment


parameters, including red and white blood cells (RBC and WBC), between all the three treated groups and the control group (Figure 7c-f). The spleen, liver, kidney, heart, and lung tissues were treated with hematoxylin and eosin (H&E) stain and examined histopathologically. The histological sections of five major organs did not show any obvious damage, inflammation, toxic side effects, or irregular anatomical structure by the treated groups, as compared to the control group (Figure 8). These results corroborated excellent biocompatibility of the CysHHC10 peptide, Au-HHC, and Au-HHC-CA NPs.


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4. Conclusion Gold nanoclusters (Au NCs) with citraconyl (CA)-protected antimicrobial peptide (AMP) coatings were fabricated via direct reduction of Au(III) by thiol-terminated CysHHC10 peptide and subsequent amidation with citraconyl anhydride. The synthesized Au-HHC-CA NCs exhibited bacterial binding and photoluminescence features, which are potentially useful for imaging and detection of bacterial infection. Under physiological condition, the Au-HHC-CA NCs showed negligible toxicity towards mouse fibroblasts as they did not record a minimal inhibitory concentration (MIC) up to 1024 µg/mL. Also, the in vivo study toward mice did not show any toxicity caused by the Au-HHC-CA NCs. This good cytocompatibility was attributed to the incorporation of Au core and anionic protection of the citraconyl moieties. However, the citraconyl amides were readily hydrolysable under acidic condition (e.g. at pH 5.5 of the bacterial infection environment) to expose the bactericidal cationic amines of the AMP coating. The bactericidal efficacy of the Au-HHC-CA NCs after hydrolysis of the citraconyl moieties was comparable to that of the CysHHC10 peptide, indicating that the antimicrobial potency of the native AMP was retained after sequential NCs formation and amidation. Thus, the Au-HHC-CA NCs presented a ‘smart’ pH-responsive and multifunctional system for antimicrobial therapy, bacterial targeting and imaging.



Associated Content The Supporting Information is available free of charge on the ACS Publications website: XPS spectra, elemental stoichiometries, MBC values, HPLC elugrams, liposomal damage, LDH leakage, mice weight monitor, and blood biochemistry analyses of the samples.

Acknowledgement This work was funded and supported by a Singapore Ministry of Education Tier 3 Grant (MOE2013-T3-1-002).


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40. Pranantyo, D.; Xu, L. Q.; Neoh, K.-G.; Kang, E.-T.; Ng, Y. X.; Teo, S. L.-M. Tea stains-inspired initiator primer for surface grafting of antifouling and antimicrobial polymer brush coatings. Biomacromolecules 2015, 16, 723-732. 41. Spampinato, V.; Parracino, M. A.; La Spina, R.; Rossi, F.; Ceccone, G. Surface Analysis of Gold Nanoparticles Functionalized with Thiol-Modified Glucose SAMs for Biosensor Applications. Front. Chem. 2016, 4. 42. Ulman, A.; Ioffe, M.; Patolsky, F.; Haas, E.; Reuvenov, D. Highly active engineered-enzyme oriented monolayers: formation, characterization and sensing applications. J. Nanobiotechnol. 2011, 9, 26. 43. Pranantyo, D.; Xu, L. Q.; Neoh, K.-G.; Kang, E.-T.; Yang, W.; Lay-Ming Teo, S. Photoinduced anchoring and micropatterning of macroinitiators on polyurethane surfaces for graft polymerization of antifouling brush coatings. J. Mater. Chem. B 2014, 2, 398-408. 44. Zheng, K.; Setyawati, M. I.; Lim, T.-P.; Leong, D. T.; Xie, J. Antimicrobial Cluster Bombs: Silver Nanoclusters Packed with Daptomycin. ACS Nano 2016, 10, 7934-7942. 45. Mansfield, E.; Tyner, K. M.; Poling, C. M.; Blacklock, J. L. Determination of Nanoparticle Surface Coatings and Nanoparticle Purity Using Microscale Thermogravimetric Analysis. Anal. Chem. 2014, 86, 1478-1484. 46. Zhou, T.; Zhou, X.; Xing, D. Controlled release of doxorubicin from graphene oxide based charge-reversal nanocarrier. Biomaterials 2014, 35, 4185-4194. 47. Pranantyo, D.; Xu, L. Q.; Kang, E.-T.; Chan-Park, M. B. Chitosan-Based Peptidopolysaccharides as Cationic Antimicrobial Agents and Antibacterial Coatings. Biomacromolecules 2018, 19, 2156-2165. 48. Pranantyo, D.; Xu, L. Q.; Kang, E.-T.; Mya, M. K.; Chan-Park, M. B. Conjugation of polyphosphoester and antimicrobial peptide for enhanced bactericidal activity and biocompatibility. Biomacromolecules 2016, 17, 40374044. 49. Garle, A. L.; Budhlall, B. M. PEG Bottle Brush Copolymers as Antimicrobial Mimics: Role of Entropic Templating in Membrane Lysis. Langmuir 2019, 35, 3372-3382. 50. Zheng, Y.; Liu, W.; Chen, Y.; Li, C.; Jiang, H.; Wang, X. Conjugating gold nanoclusters and antimicrobial peptides: From aggregation-induced emission to antibacterial synergy. J. Colloid Interface Sci. 2019, 546, 1-10. 51. Jalaei, J.; Layeghi-Ghalehsoukhteh, S.; Hosseini, A.; Fazeli, M. Antibacterial effects of gold nanoparticles functionalized with the extracted peptide from Vespa orientalis wasp venom. J. Pept. Sci. 2018, 24, e3124. 52. Fischer, D.; Li, Y.; Ahlemeyer, B.; Krieglstein, J.; Kissel, T. In vitro cytotoxicity testing of polycations: influence of polymer structure on cell viability and hemolysis. Biomaterials 2003, 24, 1121-1131.


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Biomacromolecules

Captions for Scheme, Tables and Figures

Scheme 1. Preparation of the peptide-reduced Au-HHC and citraconyl-protected AuHHC-CA NCs. Table 1. Particle diameter, zeta potential, and composition of the Au-HHC and AuHHC-CA NCs. Table 2. Antimicrobial activity, hemolytic rate, cytotoxicity, and therapeutic index of the peptide-reduced and citraconyl-protected Au NCs. Figure 1. 1H NMR spectra of the CysHHC10 peptide (black), Au-HHC (red), and AuHHC-CA NCs (blue) in D2O. Figure 2. Digital photographs of the HAuCl4 and CysHHC10 mixture before reaction (left), Au-HHC NCs (middle), and Au-HHC-CA NCs (right) under (a) visible room light and (b) 365-nm UV light. TEM images of the (c) Au-HHC NCs and (d) AuHHC-CA NCs. Scale bars are 20 nm. Figure 3. (a) Photoemission spectra under excitation wavelength of 365 nm and (b) thermogravimetric curves of the CysHHC10 (black), Au-HHC NCs (red), and AuHHC-CA NCs (blue). (c) Time-dependent degradation of citraconic amide of the AuHHC-CA NCs under different pH. (d) Relative ROS level of bacteria after incubation with the samples for 2 h. The data were normalized to bacterial cell number, and the ROS level of blank control was set to 1. Error bar denotes standard deviation obtained from three replicates. Asterisk (*) and hash (#) denote significant difference toward blank sample of the respective bacteria, with p-value < 0.05 (Tukey’s test).



Figure 4. (a) Antimicrobial activity of the Au-HHC-CA NCs towards Gram-negative and Gram-positive bacteria under different pH. (b) DiSC3-5 release and NPN uptake on E. coli and S. aureus after incubation with the samples in PBS for 1 h. Error bar denotes standard deviation obtained from three replicates. Figure 5. Flow cytometric histograms of (a) E. coli and (b) S. aureus on 525-nm UV filter. Digital photographs of (c) E. coli and (d) S. aureus sediments under 365-nm UV irradiation after being incubated with the (i) CysHHC10 peptide, (ii) Au-HHC, (iii) Au-HHC-CA pretreated in pH 7.4, and (iv) Au-HHC-CA NCs pretreated in pH 5.0. The NCs and bacterial concentrations are 50 µg/mL and 108 cells/mL, respectively. Figure 6. Fluorescence micrographs of the bacterial-adhered (a,d) Au-HHC, (b,e) AuHHC-CA pretreated at pH 5.0, and (c,f) Au-HHC-CA NCs pretreated at pH 7.4 after incubation in the (a–c) E. coli and (d–f) S. aureus suspensions at 37 °C for 3 h. The NCs and bacterial concentrations were 50 µg/mL and 108 cells/mL, respectively. Scale bar is 20 µm. Figure 7. (a) Hemolytic activity of the samples after incubation with rat whole blood for 1 h and (b) relative cell viability of the 3T3 fibroblasts after incubation with the samples for 24 h, with error bar denotes standard deviation obtained from three replicates. Counts of (c) red blood cells (RBC), (d) white blood cells (WBC), (e) platelets (PLT), and (f) hemoglobin (HGB) concentration after 7 days post i.p. injection of 5 mg/kg of the samples to mice. Blood parameters from each mouse were plotted as individual points, and error bar represents standard deviation within an experimental group of 5 mice. Figure 8. Representative H&E stained images of the spleen, liver, kidney, heart and lung collected from mice sacrificed after 7 days post i.p. injection of 5 mg/kg of the samples (magnification: 200×).


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Biomacromolecules

Scheme 1. Preparation of the peptide-reduced Au-HHC and citraconyl-protected AuHHC-CA NCs

NH2

HAuCl4

+

NH2

O

H 2N

N H

HS

O

H N

H N

N H

O

NH O

NH N H

O

NH HN

CysHHC10

S

O NH

NH2

N H

O NH

O

NH H 2N

NH

NH2

O N H

N H

O

H N

NH2

NH2

H 2N

O

H N

NH H O

O

H N

N

N H

O

N H

O

NH HN

NH O

H N

N H

O NH

H N

O

O NH

NH2

O

NH H 2N

Au-HHC

NH2

N H

NH

O O O CA

HO

HN HO

HO

O O

HN

O O N H S

O N H

H N

O

NH H O

O

N

N H

O

O

O

NH N H

NH HN

Au-HHC-CA

NH

HO

H N O NH

O N H

H N

O

O NH

O

O

O

ACS Paragon Plus Environment

O O

NH

O

35

H N

N H

HN

OH

NH

O

OH


Page 36 of 47

Table 1. Particle diameter, zeta potential, and composition of the Au-HHC and AuHHC-CA NCs

Sample

Particle diameter a (nm)

Zeta potential, ζ

Organic fraction b

TEM

DLS

(mV)

(%-mass)

-

-

21.5 ± 2.3

100

Au-HHC

2.4 ± 0.6

4.2 ± 1.1

31.4 ± 5.7

83

Au-HHC-CA

2.6 ± 0.7

6.8 ± 1.5

-42.3 ± 4.6

89

CysHHC10

a

Dry-state and hydrodynamic diameters of the NCs were determined from TEM image and DLS measurement, respectively.

b

Organic mass-fraction of the NCs was determined from the TGA analysis.


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Biomacromolecules

Table 2. Antimicrobial activity, hemolytic rate, cytotoxicity, and therapeutic index of the peptide-reduced and citraconyl-protected Au NCs

MIC, µg/mL a

Selectivity b

E. coli

P. aeruginosa

S. aureus

S. epidermidis

HC50 µg/mL

32

64

32

8

670

> 43

>1

>1

>1

>5

Au-HHC

16 (13)

32 (27)

16 (13)

4 (3)

765

> 56 (47)

>4

>2

>4

> 14

Au-HHC-CA, pH 7.4

>> 512 c

>> 512 c

>> 512 c

>> 512 c

>> 1024 c

>> 1024 c

-

-

-

-

Au-HHC-CA, pH 5.0

32 (26)

64 (53)

32 (27)

8 (7)

-

> 102 (85)

>3

>2

>3

> 13

Sample CysHHC10

IC50 µg/mL

E. coli

P. aeruginosa

S. aureus

S. epidermidis

a

Values in bracket express the mass of peptide only in the NCs, calculated based on the peptide mass fraction.

b

Selectivity values were calculated as IC50/MIC.

c

The values were not observed up to the highest concentrations of compound tested (512 µg/mL for MIC, 1024 µg/mL for HC50 and IC50).



Figure 1. 1H NMR spectra of the CysHHC10 peptide (black), Au-HHC (red), and AuHHC-CA NCs (blue) in D2O.


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Biomacromolecules

Figure 2. Digital photographs of the HAuCl4 and CysHHC10 mixture before reaction (left), Au-HHC NCs (middle), and Au-HHC-CA NCs (right) under (a) visible room light and (b) 365-nm UV light. TEM images of the (c) Au-HHC NCs and (d) AuHHC-CA NCs. Scale bars are 20 nm.

a

c

b

d



Figure 3. (a) Photoemission spectra under excitation wavelength of 365 nm, and (b) thermogravimetric curves of the CysHHC10 (black), Au-HHC NCs (red), and AuHHC-CA NCs (blue). (c) Time-dependent degradation of citraconic amide of the AuHHC-CA NCs under different pH. (d) Relative ROS level of bacteria after incubation with the samples for 2 h. The data were normalized to bacterial cell number, and the ROS level of blank control was set to 1. Error bar denotes standard deviation obtained from three replicates. Asterisk (*) and hash (#) denote significant difference toward blank sample of the respective bacteria, with p-value < 0.05 (Tukey’s test).

a

b

d


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Biomacromolecules

c



Figure 4. (a) Antimicrobial activity of the Au-HHC-CA NCs towards Gram-negative and Gram-positive bacteria under different pH. (b) DiSC3-5 release and NPN uptake on E. coli and S. aureus after incubation with the samples in PBS for 1 h.

a

b


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Biomacromolecules

Figure 5. Flow cytometric histograms of (a) E. coli and (b) S. aureus on 525-nm UV filter. Digital photographs of (c) E. coli and (d) S. aureus sediments under 365-nm UV irradiation after being incubated with the (i) CysHHC10 peptide, (ii) Au-HHC, (iii) Au-HHC-CA pretreated in pH 7.4, and (iv) Au-HHC-CA NCs pretreated in pH 5.0. The NCs and bacterial concentrations are 50 µg/mL and 108 cells/mL, respectively.



Figure 6. Fluorescence micrographs of the bacterial-adhered (a,d) Au-HHC, (b,e) AuHHC-CA pretreated at pH 5.0, and (c,f) Au-HHC-CA NCs pretreated at pH 7.4 after incubation in the (a–c) E. coli and (d–f) S. aureus suspensions at 37 °C for 3 h. The NCs and bacterial concentrations were 50 µg/mL and 108 cells/mL, respectively. Scale bar is 20 µm.

a

d

b

e

c

f


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Biomacromolecules

Figure 7. (a) Hemolytic activity of the samples after incubation with rat whole blood for 1 h and (b) relative cell viability of the 3T3 fibroblasts after incubation with the samples for 24 h, with error bar denotes standard deviation obtained from three replicates. Counts of (c) red blood cells (RBC), (d) white blood cells (WBC), (e) platelets (PLT), and (f) hemoglobin (HGB) concentration after 7 days post i.p. injection of 5 mg/kg of the samples to mice. Blood parameters from each mouse were plotted as individual points, and error bar represents standard deviation within an experimental group of 5 mice.

a

b

c

d

e

f



Figure 8. Representative H&E stained images of the spleen, liver, kidney, heart and lung collected from mice sacrificed after 7 days post i.p. injection of 5 mg/kg of the samples (magnification: 200×).


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TOC Graphic

Title: Antimicrobial Peptide-reduced Gold Nanoclusters with Charge-reversal Moieties for Bacterial Targeting and Imaging

Authors: Dicky Pranantyo, Peng Liu, Wenbin Zhong, En-Tang Kang, and Mary B. Chan-Park

Summary/highlights: Gold nanoclusters functionalized with cationic peptide and citraconyl moieties are photoluminescent and cytocompatible in physiological condition, with on-demand switch into microbial targeting and bactericidal mode in acidic condition.


Antimicrobial Peptide-Reduced Gold Nanoclusters with Charge

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