Structure-Dependent Antimicrobial Theranostic Functions of Self

Specific cell-targeted drug delivery using biocompatible nanomaterials that enhance bioavailability and reduce systemic toxicity is attracting interes...
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Structure-Dependent Antimicrobial Theranostic Functions of Self-Assembled Short-Peptide Nanoagents Inhye Kim, Seon-Mi Jin, Eun Hee Han, Eunhee Ko, MiJa Ahn, Woo-Young Bang, Jeong Kyu Bang, and Eunji Lee Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b00951 • Publication Date (Web): 24 Aug 2017 Downloaded from http://pubs.acs.org on August 24, 2017

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Structure-Dependent Functions

of

Antimicrobial

Self-Assembled

Theranostic Short-Peptide

Nanoagents Inhye Kim,† Seon-Mi Jin,† Eun Hee Han,‡,ǁ Eunhee Ko,† MiJa Ahn,┴ Woo-Young Bang,† Jeong-Kyu Bang,*,§,# and Eunji Lee*,† †

Graduate School of Analytical Science and Technology, Chungnam National University, Daejeon 34134, Republic of Korea ‡

Division of Bioconvergence Analysis, §Division of Magnetic Resonance, Korea Basic Science Institute, Cheongju 28119, Republic of Korea ǁ

Immunotherapy Convergence Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 34141, Republic of Korea

┴

Anticancer Agent Research Center, Korea Research Institute of Bioscience and Biotechnology, Cheongju 28116, Republic of Korea #

Department of Bio-analytical Science, University of Science & Technology, Daejeon 34113, Republic of Korea.

Abstract

Gadolinium (Gd[III])-based nanoaggregates are potential noninvasive magnetic resonance imaging (MRI) probes with excellent spatial and temporal resolution for cancer diagnosis. Peptides conjugated with Gd3+ can aid in supramolecular scaffolding for MRI nanoagents because of their inherent biocompatibility and degradability. We report here a strategy to tune the MR relaxivity of tumor-cell targeted nanoagents and enhance the antimicrobial and anticancer activities of nanoagents based on rationally designed antimicrobial peptide (AMP) assembly. A tripeptide with glycyl-L-histidyl-L-lysine ACS Paragon Plus Environment

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(GHK) capable of Gd3+-chelation was attached to short AMPs containing pyrazole amino acids that spontaneously assembled as a function of the number of hydrophobic amino acid residues and the peptide length of AMPs. Aqueous co-assembly of GHK with tumor-targeting, cyclic arginine-glycineaspartic acid (cRGD)-tagged AMPs resulted in the formation of micelles, fibrils, vesicles, sheets, and planar networks. Interestingly, the two-dimensional planar network nanostructure showed less antibacterial activity and tumor-cell cytotoxicity, but greater drug loading/delivery and magnetic resonance signaling than micelles because of its intrinsic structural characteristics. This study can provide the rational approach for design and fabrication of clinically useful nanoagents.

Keywords: self-assembly, antimicrobial peptide, nanostructure, magnetic resonance imaging, specific cell-targeting, theranostics

1. Introduction Specific cell-targeted drug delivery using biocompatible nanomaterials that enhance bioavailability and reduce systemic toxicity is attracting interest for clinical applications.1 Associating an imaging modality with a therapeutic vehicle is a useful strategy for the development of a diagnostic nanoagent for optimized, personalized treatment of diseases.2 Several supramolecular scaffolds for theranostic agents have been proposed, including bioactive peptides, dendrimers, proteins, lipids, and polymers.3-6 Among these molecular scaffolds, peptides are the most exciting platforms because of their excellent biocompatibility, biodegradability, structural versatility, and ease of chemical modification.7-9 In particular, in aqueous solution, peptide with amphiphilic characteristic can form into various, selfassembled core-shell nanostructures, wherein the core provides a cavity for hydrophobic drugs and the shell allows particle stabilization and functional modification by adopting a bioactive moiety, addressing current clinical challenges.10 Depending on their shape and size, peptide nanostructures offer advantages in stability against enzymatic degradation, encapsulation ability of drugs, sustained drug release, and adjuvanting properties, as therapeutic agents.10,11

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Meanwhile, paramagnetic gadolinium (Gd[III])-conjugated scaffolds have been used as magnetic resonance imaging (MRI) contrast agents that enable noninvasive monitoring of their in vivo distribution with excellent anatomical and temporal resolution. Many efforts have been devoted to increasing their sensitivity, which requires injecting high concentrations of the agents.12 The MR efficacy of a nanoagent estimated by paramagnetic enhancement of the longitudinal water proton relaxation rate could be improved by structural modifications affecting parameters such as hydration number, water exchange rate, and rotational dynamics.13 Therefore, supramolecular nanoaggregates with structural diversity has been recognized as an attractive platform for tuning MR efficacy.7 Interest in supramolecular MRI contrast nanoagents based on amphiphilic peptide self-assembly has been growing since they were first developed by Stupp and Meade,14 when macrocylic 1,4,7,10tetraazacyclododecane-1,4,7,10-tetraacetic acids (DOTAs) for anchoring Gd3+ chelates were conjugated to peptides. The formation of supramolecular aggregates with certain molecular weights allowed an increase in rotational correlation time (τr) that enhanced relaxivity.15 Relaxivity was further controlled by changing the morphology of nanoaggregates for different physical properties that were dependent on the peptide design and assembly environment.14,16,17 More precisely, the closer positioning of Gd3+ chelates to the hydrophobic end of peptide amphiphiles has been reported to decrease internal flexibility and increase steric hindrance of Gd3+ chelates, resulting in higher relaxivity.18 Therefore, the attachment of a bulky macrocyclic moiety such as DOTA or diethylenetriamine pentaacetic acid (DTPA), mostly for chelating Gd3+, has been recognized as one of key factors determining the molecular packing arrangement due to their steric hindrance, which affects MR efficacy. A more convenient strategy to prepare Gd3+-chelating nanoaggregates without a macrocyclic moiety as well as to control the nanostructures is therefore desirable. Recently, cationic, amphiphatic antimicrobial peptides (AMPs) have been regarded as a promising new generation of antibiotics because of their rapid and broad-spectrum antimicrobial properties, their ability to kill multidrug-resistant bacteria, and their low propensity to develop resistance.19,20 In contrast to the self-assembled peptide antibacterial nanoagents imposed by chelation of antibacterial metal ions, ACS Paragon Plus Environment

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such as Ag+, Zn2+, and Cu2+,21,22 AMPs could overcome undesirable side effects including oxidative DNA damages and immune responses to human body. It has also been known that some AMPs can exhibit cytotoxic activity against cancer cells.23 The hydrophobic and electrostatic interactions between negatively charged bacterial or cancer cells and positively charged AMPs are believed to significantly increase binding strength and selectively disrupt biomembranes.24,25 Therefore, we anticipated that the utilization of AMPs as a molecular scaffold for the fabrication of theranostic nanoagents could not only endow antimicrobial activity, but also overcome the antibacterial and anticancer resistance of nanoagents.22 Along this line, two peptides based on AMPs consisting of N-alkyl/aryl pyrazole (Py) amino acid derivatives and arginine (Arg) were designed (Figure 1).26 MRI and specific tumor-targeting properties were conferred by the addition of tripeptide glycine-histidine-lysine (GHK) for Gd3+-decoration of the shells of aggregates upon self-assembly27 and the addition of cyclic arginine-glycine-aspartic acid (cRGD) for the recognition of αvβ3 and αvβ5 integrin receptors expressed on tumor cells.28 GHK peptide is also known to show antimicrobial activity.29 Herein, we report on short peptide-based cancertargeting, theranostic nanoagents with antimicrobial activity. Their MRI contrast characteristics were investigated as a function of nanoagent morphology related to the number of hydrophobic Py residues and the repeating Py-Arg building units of AMPs (Figure 1). Notably, the two-dimensional (2D) network nanoagent formed by robust hydrophobic interactions of AMPs showed lesser antimicrobial activity and tumor-cell cytotoxicity than that of spherical aggregates, but more drug-encapsulation capacity and greater longitudinal (T1) relaxivity of water protons because of its intrinsic structural properties. Therefore, the physical properties of aggregates such as size, shape and charge should be considered when theranostic nanoagents are fabricated.

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Figure 1. Schematic illustration of the fabrication of a theranostic agent by co-assembly of short AMPs.

2. Experimental 2.1. Materials 4-Nitro-3-pyrazolecarboxylic

acid,

thionyl

chloride,

anhydrous

1-(2-bromoethyl)naphthalene,

gadolinium(lll)

chloride

(GdCl3)

powder,

and

dihydrochloride

(DAPI),

4-(2-hydroxyethyl)piperazine-1-ethanesulfonic

Nile

Red,

4’,6-diamidine-2’-phenylindole acid

(HEPES)

buffer,

paraformaldehyde, trifluoroacetic acid (TFA, 99.0%), and Triton X-100 were purchased from SigmaAldrich. Sodium bicarbonate (NaHCO3), sodium sulfate (Na2SO4), brine, potassium carbonate (K2CO3), dicholoromethane (DCM, 99.5%), piperidine (99.0%), nitric acid, acetone (99.5%), and hydrochloric acid, and magnesium sulfate (MgSO4) were obtained from Daejung Chemicals & Metals. All protected amino acids, Rink amide 4-methylbenzhydrylamine (MBHA) resin and Wang resin were purchased from Calbiochem-Novabiochem (La Jolla, CA, USA). Protected cyclic RGDyK(NH2) (cRGD) was ACS Paragon Plus Environment

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obtained from FutureChem Co. LTD (Seoul, Republic of Korea). 2-(1H-benzotriazole-1-yl)-1,1,3,3tetramethyluronium hexafluorophosphate (HBTU), N,N-diisopropylethylamine (DIEA, 99.0%), methyl phenyl sulfide (99.0%), 1,2-ethanedithiol (98.0%), and hydroxybenzotriazole (HOBt) were used as received from Merck. N,N-Dimethylformamide (DMF, 99.5%) from Fisher was used. Py amino acid (4[(9-fluoren-9-ylmethoxy)carbonylamino]-1-[2-(naphthalen-1-yl)ethyl]-1H-pyrazole-3-carboxylic

acid)

was synthesized according to the experimental procedure reported in literature (Figure S1).26 Doxorubicin hydrochloride (DOX·HCl, Boryung Pharmaceutical Co., Ltd., Seoul, Republic of Korea) was desalinated before use. MCF-10A cells, HeLa cells and NIH3T3 cells were obtained from American Type Culture Collection (Manassas, VA, USA). Trypsin was purchased from Life Technologies, Inc. (Carlsbad, CA, USA). 2.2. Synthesis of G1 G1 was prepared by using Rink amide MBHA resin with an initial loading of 0.61 mmol/g by swelling in DMF for 45 min prior to initiate the synthesis. For sequence extension, initially, the Fmoc-Arg(Pbf)OH (5.0 equiv.) along with HBTU (5.0 equiv.), HOBt (5.0 equiv.) in DMF (2 mL) was added to the free amine on resin in the presence of DIEA (5.0 equiv.) and the coupling was allowed to proceed for 1 h with vortex stirring. Then the resin was washed with DMF and the deprotection of Fmoc was achieved by treating with 20% piperidine in DMF (1 x 10 min, 2 x 3 min). The resin was washed again, and the same deprotection/condensation procedure was repeated for the successive introduction of Py amino acid, Boc-Lys(Mtt)-OH. Further to extend the sequence, the protecting group (Mtt) was cleaved by treating with 1% TFA in DCM and washed with DMF, to which Fmoc-His(Trt)-OH along with HBTU (5.0 equiv.), HOBt (5.0 equiv.) were added to enable the coupling. Further, resin was treated with 20% piperidine in DMF to cleave the Fmoc group and subsequently coupled with Boc-Gly-OH under the prescribed condition. Finally, the resin was adopted the successive washing of DMF, DCM, and ether, and then dried under vacuum (Figure S2).

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2.3. Synthesis of G2 Solid phase peptide synthesis (SPPS) of G2 was achieved by following the procedure of G1 using the amino acid sequences, Fmoc-Arg(Pbf)-OH, Py amino acid (two subsequent coupling), and BocLys(Mtt)-OH. After the Mtt cleavage, Fmoc-His(Trt)-OH and Boc-Gly-OH were coupled successively. 2.4. Synthesis of G3 SPPS of G3 was achieved by following the procedure of G1 using the amino acids sequences, FmocArg(Pbf)-OH, Py amino acid, Fmoc-Arg(Pbf)-OH, Py amino acid, and Boc-Lys(Mtt)-OH. After the Mtt cleavage, further sequence extension was carried out by coupling with Fmoc-His(Trt)-OH and Boc-GlyOH, respectively. 2.5. Cleavage of G1-G3 Simultaneous deprotection and cleavage of the peptide from the resin were conducted by treating of TFA, water, and triisopropylsilane (90:5:5, v/v/v, 2 mL) for 2 h. The crude peptide was precipitated from the cleavage mixture by addition of cold diethyl ether. Purification of crude peptide was carried out on the preparative Vydac C18 column using an appropriative 10-90%/30 min water/acetonitrile gradient in the presence of 0.05% TFA. Purified peptides (> 95%) were assessed by reverse-phase-high performance liquid chromatography (RP HPLC) on an analytical Vydac C18 column (Figure S3). Peptides were identified using matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI-TOF/TOF-MS) (Bruker Daltonik GmbH, Bremen, Germany) (Figure S4). G1: MS (MALDI-TOF/TOF) m/z = 760 [M+H]+, G2: MS (MALDI-TOF/TOF) m/z = 1023 [M+H]+, 1045 [M+Na]+, G3: MS (MALDI-TOF/TOF) m/z = 1179 [M+H]+. 2.6. Synthesis of R1 Wang resin (0.61 mmol) was swollen in DCM/DMF (8:2) for 1.5 h, and the first amino acid, FmocArg(Pbf)-OH (5.0 equiv.) was coupled to the resin using diisopropylcarbodiimide (DIC, 5.0 equiv.), and ACS Paragon Plus Environment

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dimethylaminopyridine (DMAP, 0.1 equiv.) in DCM/MeOH (8:2) and allowed to stir for 20 h. The Fmoc group was cleaved with piperidine in DMF (20%). The other amino acids, including Py amino acid, Fmoc-Glu(OAll)-OH were coupled successively using HBTU (5.0 equiv.), HOBt (5.0 equiv.) in DMF (2 mL) in the presence of DIEA (5.0 equiv.). To extend the peptide chain, O-allyl group of glutamic acid was deprotected using Pd(PPh3)4 (4.0 equiv.) in chloroform (CHCl3):acetic acid (AcOH):N-ethylmaleimide (NEM) (37:2:1) while stirring for 1.5 h. The resin was washed with DCM, THF, and DMF. Then the free amino acid was coupled with protected cRGD using HBTU (5.0 equiv.), HOBt (5.0 equiv.) in DMF and stirred for 1 h. The resin was treated with 20% piperidine in DMF to cleave the Fmoc group (Figure S5). 2.7. Synthesis of R3 Wang resin (0.61 mmol) was swollen in DCM/DMF (8:2) for 1.5 h, and the first amino acid, FmocArg(Pbf)-OH (5.0 equiv.) was coupled to the resin using DIC (5.0 equiv.), and DMAP (0.1 equiv.) in DCM/MeOH (8:2) and allowed to stir for 20 h. The Fmoc group was cleaved with piperidine in DMF (20%). The other amino acids, including Py amino acid, Fmoc-Glu(OAll)-OH were coupled successively using HBTU (5.0 equiv.), HOBt (5.0 equiv.) in DMF (2 mL) in the presence of DIEA (5.0 equiv.). To extend the peptide chain, O-allyl group of glutamic acid was deprotected using Pd(PPh3)4 (4.0 equiv.) in CHCl3:AcOH:NEM (37:2:1) while stirring for 1.5 h. The resin was washed with DCM, THF, and DMF. Then the free amino acid was coupled with protected cRGD using HBTU (5.0 equiv.), HOBt (5.0 equiv.) in DMF and stirred for 1 h. The resin was treated with 20% piperidine in DMF to cleave the Fmoc group. 2.8. Cleavage of R1, R3 Finally, deprotection and cleavage from the resin were performed with a DCM/TFA mixture (1:1) for 2 h. The crude peptide was precipitated from the cleavage mixture by addition of cold diethyl ether and purification of crude peptide was carried out on the preparative Vydac C18 column using an ACS Paragon Plus Environment

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appropriative 10-90%/30min water/acetonitrile gradient in the presence of 0.05% TFA. Purified peptides (> 95%) were assessed by RP HPLC on an analytical Vydac C18 column. Mass spectra of peptides were obtained using MALDI-TOF/TOF-MS (Figures S3 and S4). R1: MS (MALDI-TOF/TOF) m/z = 1168 [M+H]+, 1190 [M+Na]+, R3: MS (MALDI-TOF/TOF) m/z = 1588 [M+H]+, 1610 [M+Na]+. 2.9. Cryogenic transmission electron microscopy (cryo-TEM) Cryo-TEM experiments were performed with a thin film of aqueous solution of sample (3 µL) transferred to a lacey supported grid by plunge-dipping method. The thin aqueous films were prepared at ambient temperature and with humidity of 97-99% within a custom-built environmental chamber in order to prevent evaporation of water from the sample solution. The excess liquid was blotted with filter paper for 2-3 sec, and the thin aqueous films were rapidly vitrified by plunging them into liquid ethane (cooled by liquid nitrogen) at its freezing point. The specimen was observed with a JEM-1400 operating at 120 kV. The data were analyzed with imaging software Simple Measure (JEOL, Tokyo, Japan) and RADIUS (Olympus Soft Imaging Solutions GmbH, Münster, Germany). 2.10. Preparation of Gd3+-complexed co-assembled AMPs (co-AMPs) The mixtures of GHK-AMP peptides and cRGD-AMP peptides were dissolved in deionized water to give a solution with concentration of 0.1 mM. GdCl3 (5.0 equiv.) was added to the co-AMPs solution at room temperature. The reaction solution was stirred for over 24 h. The aqueous solution was put into a dialysis tube (molecular weight cut-off 1,000 Da) and subjected to dialysis for 10 h under dark with stirring. 2.11. Bacteria strain and cultivation Two types of bacteria were employed: Escherichia coli (E. coli) ATCC8739 as the Gram-negative model, and Staphylococcus aureus (S. aureus) ATCC6538p as the Gram-positive model. The E. coli ATCC8739 were grown in Luria Burtani (LB) medium supplemented with erythromycin (300 µg/ml) at ACS Paragon Plus Environment

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37 °C and 200 rpm, and the S. aureus ATCC6538p were grown in pure LB medium. After overnight incubation, bacteria were harvested by centrifugation at 5,000 rpm, and bacterial pellet was resuspended with fresh LB media to conduct batch experiment. 2.12. LB liquid medium turbidity assay for antibacterial activity E. coli ATCC8739 and S. aureus ATCC6538p were prepared in sterilized glass tube, respectively. Turbidity assay was conducted with turbidity reader (kit DensiCHEK™ Plus Standards, Bio Merieux, USA) under 25 µM of each Gd3+-complexed co-AMPs sample at each time point and with Ampicillin (100 µg/mL) as a positive control. Values were gained after setting only LB broth as a blank. 2.13. Bacteria kinetic test A starter culture of each strain was inoculated with fresh colonies and incubated for 14 h overnight in LB media. Fresh media were inoculated with the starter culture and grown to an OD600 of 0.1 at 37 °C with continuous agitation at 200 rpm. LB broth containing bacteria was used as a positive control. Bacterial growth behaviors were determined by measuring the optical density at 600 nm at desired time point after treatment of Gd3+-complexed co-AMPs using a microplate reader (Spectramax M4, Molecular Devices, USA). 2.14. Hemolytic activity30 Hemolysis assays were conducted to assess the preliminary toxicity of the Gd3+-complexed co-AMPs (Gd3+-G1R3 and Gd3+-G3R3) to mouse red blood cells (mRBC) as representative mammalian cells. Fresh blood (200 µL) was washed with sterile HEPES buffer (12 mL) and centrifuged at 3,000 rpm, followed by resuspension of the pellet into 20 mL sterile HEPES buffer. The Gd3+-complexed co-AMPs (40 µL) were incubated with mRBC stock suspension (160 µL) in sterile HEPES buffer at 37 °C for 1 h with a peptide concentration ranging from 25 µM, 50 µM to 100 µM. The 100% hemolytic positive control was the HEPES buffer containing 1% Triton X-100. After 1 h incubation, the mixtures were iceACS Paragon Plus Environment

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cooled and centrifuged for 5 min at 3,000 rpm at 4 °C. The supernatant (100 µL) was added to the wells of a 96-well microplate. The experiments were run in triplicate and the percent hemolysis was determined by measuring the absorbance in the supernatant at a wavelength of 450 nm (Spectramax M4, Molecular devices, USA) using the following formula. Hemolysis (%) = [(AP – AB)/(AC – AB)] x 100, where AP is the absorbance value for a known Gd3+complexed co-AMP concentration, AC is the absorbance value for the Triton X-100 positive control, AB is the absorbance value for HEPES buffer. 2.15. Cell viability assay Cells were cultured in 96-well plates at a density of 1 x 104 cells/well. Gd3+-complexed co-AMPs were added to each individual well and then incubated at 37 °C for 12 h. The Cell Counting Kit-8 (CCK-8, Dojindo Molecular Technologies, Rockville, ML, USA) solution was added (10 µL per well), and the plates were incubated for 1 h at 37 °C. Cell viability was measured in triplicate using a CCK-8 according to the manufacturer’s protocol. 2.16. Preparation of Nile Red-encapsulated nanostructures 2.5 µL of Nile Red (12 mM) dissolved in acetone was added to 200 µL of Gd3+-complexed co-AMP solution and then vortexed and sonicated. The acetone was allowed to evaporate by opening the vial cap overnight. The solution was then lyophilized to remove any remained acetone. The dried residues were redissolved in phosphate buffered saline (PBS) buffer. The absorption spectra obtained from NEOSYS2000 spectrometer (Scinco, Seoul, Republic of Korea) at 10 mm of path length in the range of 200-900 nm. The emission spectra obtained from FS-2 fluorescence spectrometer (Scinco, Seoul, Republic of Korea) of Gd3+-complexed co-AMP solution and Nile Red-encapsulated, Gd3+-complexed co-AMP solution were compared (10 mm of path length, λex = 550 nm). 2.17. Preparation of DOX-encapsulated nanostructures ACS Paragon Plus Environment

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The DOX·HCl was first dissolved in deionized water, followed by addition of triethylamine (TEA) with the molar ratio of DOX:TEA = 1:3 to afford free DOX solution. The mixture was vigorously stirred overnight under dark condition. The prepared hydrophobic DOX in acetone was introduced into the Gd3+-complexed co-AMP solution with the molar ratio of DOX to AMP 3:1 and then sonicated. The acetone was evaporated under reduced pressure and then distilled water was added to afford 0.1 mM of Gd3+-complexed co-AMP solution. The aqueous solution was put into a dialysis tube (molecular weight cutoff 1,000 Da) and subjected to dialysis for 10 h under dark with stirring. The distilled water was replaced every 2 h to remove unloaded drug. Samples were lyophilized and reconstituted in distilled water for drug-loading ability test with spectroscopic analysis. 2.18. Analysis of cellular uptake by confocal laser scanning microscopy (CLSM) To demonstrate the cellular uptake of Gd3+-complexed co-AMPs, cells were seeded in confocal slide at 40,000 cells/well in complete cell culture media. Cell imaging plates were acquired from Ibidi GmbH (Ibidi, Munich, Germany). The cells were treated with Nile Red-loaded, Gd3+-complexed co-AMPs at indicated concentrations for desired time, cellular images of intracellular localization were obtained by an inverted CLSM. Images were obtained with the ZEN2009 software (Carl Zeiss, Oberkochen, Germany). To stain the nucleus, cells were fixed with 4% paraformaldehyde for 5 min, rinsed 3 times with PBS and then incubated for 10 min with DAPI solution. Cells were mounted in polyvinylalcohol mounting medium with DABCO® (Sigma). 2.19. Flow cytometry analysis Cells were seeded in 12-well plates and treated with Gd3+-complexed co-AMPs for 1 h. After that, medium was removed and cells were harvested by trypsin and then fixed by 4% paraformaldehyde and washed several times with PBS. Cells were analyzed by using flow cytometer (MoFlo Astrios, Beckman Coulter, Miami, USA). Laser excitation and emission band pass wavelengths were 488 nm and 576 nm, respectively. The results are reported as the median of the distribution of cell fluorescence intensity ACS Paragon Plus Environment

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obtained by analyzing 10,000 cells in the gate. The results are analyzed by Summit Software (Version 6.0, Beckman Coulter, Miami, USA). Values of the internalization score and mean fluorescence intensity (MFI) were calculated for at least 5,000 cells per sample.

2.20. Quantification of paramagnetic Gd3+complexed to the co-assembled nanostructures Samples for analysis were prepared by taking 200 µL from the stock solution used for relaxivity and T1 measurements and placed in vial filled with nitric acid and hydrochloric acid with a volume ratio of 3:1. The solution was ultrasonicated for overnight and then digested at 110 °C for 2 h. Samples were diluted in deionized water (10 mL) for inductively coupled plasma-mass spectrometry (ICP-MS) analysis. Calibration was conducted with 0-50 ppb of Gd3+ standards.

3. Results and discussion 3.1. Synthesis and self-assembly of AMPs In designing peptides with antimicrobial activities, controlling hydrophobicity and amphiphaticity of peptides is critical. We designed a series of AMPs consisting of Py and Arg building blocks (Figure 2), with GHK for Gd3+ chelating (G1-G3) and cRGD for tumor-targeting (R1 and R3). The Py and Arg building blocks were responsible for maintaining the hydrophobicity and positive charge of the AMP. The unpaired electron in GHK served as coordination sites for Gd3+ (Figure S6).27,31 AMPs G1 and R1 with alternating Py and Arg residues were synthesized based on Fmoc (9-fluorenylmethoxycarbonyl) chemistry with SPPS. Each GHK and cRGD was covalently attached to the N-terminus of Py-Arg (Figures S2 and S5). An additional Py amino acid was introduced between Py and Arg of G1 to enhance hydrophobic interactions for self-assembly of AMPs, and the resulting structure was named G2. G3 and R3 were obtained by repeating Py-Arg building units to create a molecular structure with increased ACS Paragon Plus Environment

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peptide length. The resulting crude product was purified by HPLC to provide pure AMPs (>95% as determined by analytical HPLC, Figure S3), and the molecular weight of each AMP was verified by MALDI-TOF/TOF-MS in comparison with a theoretical value (Figure S4). The newly synthesized AMPs were also characterized by 1H nuclear magnetic resonance (NMR) analysis (Figure S7).

Figure 2. Molecular structures of AMPs with metal-chelating moieties a) G1, b) G2, and c) G3, and with cell-targeting moieties d) R1 and e) R3. Red: Arg; black: hydrophobic Py amino acid residue; green: cRGD; and blue: GHK. To investigate the self-assembly behavior of the AMPs, dynamic light scattering (DLS) and TEM measurements were performed (Figure S8 and Figure 3). TEM images confirmed the rationally designed molecular structure-dependent tuning of the morphology of the nanostructures formed in aqueous solution. G1 shows the formation of spherical micellar structures with a diameter of 5-6 nm (Figure 3a), which is consistent with the size determined by DLS (Figure S8). Interestingly, G2 containing an additional Py residue formed tubular structures with a wall thickness of ~6 nm (Figure 3b). A TEM image of G2 negatively stained with 2 wt% uranyl acetate solution showed the presence of a vacant internal channel (inset of Figure 3b). The staining agent easily penetrated the hollow cylinder by ACS Paragon Plus Environment

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capillary force. This structural transformation can be explained by the enhanced hydrophobic interactions between neighboring G2 imposed by attaching an additional Py. The emission spectra of G2 primarily exhibited the excimer-like band of a naphthalene ring chromophore, whereas G1 showed the excimer band along with a monomer-like band (λex = 280 nm, Figure S9).32 The ratio of excimer to monomer emissions increased with the addition of Py to G1, indicating that hydrophobic interactions increased upon self-assembly and with close proximity between the naphthalene rings.33 This result is consistent with the critical micellar concentration (CMC) values of G1 and G2. The CMCs of AMPs were measured by fluorescence spectroscopy using Nile Red (Figure S10).34 When aggregates form in aqueous solution, Nile Red having a very poor water solubility can be encapsulated within the hydrophobic core, resulting in the characteristic peak of Nile Red in the emission spectra.35 As expected, G2 showed a lower CMC than G1 (~20 µM for G1 and ~16 µM for G2, respectively), indicating that G2 has stronger hydrophobic interactions during aqueous self-assembly. The synergetic effect of these strengthened hydrophobic interactions and enhanced molecular stiffness by the addition of Py to G1 would drive the formation of a ribbon-like structure, which in turn resulted in a tubular structure by coiling to minimize entropically unfavorable contacts between hydrophobic block and water.36 Indeed, the ribbon-like structure was observed at the early stage of self-assembly of G2 (Figure S11c). Interestingly, G3, composed of two Py and two Arg residues arranged alternately, formed a vesicular structure ranging 30-70 nm in size (Figure 3c). An increase in peptide length lowers the CMC value (Figure S10) and sandwich-like packing stiffness by facial stacking of AMPs, which affects the curvature of aggregates relative to that of G2 and results in the formation of vesicles rather than tubules.37,38 R1 with a cRGD pendant, which had a structure similar to that of G1, formed a ribbon-like structure (Figure 3d). The presence of a macrocycle peptide capable of hydrogen bonding can enhance the intermolecular facial interactions between neighboring AMPs,39,40 resulting in the formation of 2D ribbon-like structures instead of spherical micelles. The increased number of repeating Py-Arg from R1 to R3 showed a morphological transition from a ribbon-like structure to a vesicular structure (Figure 3e). Cryo-TEM was performed to confirm the presence of vesicular structures in aqueous solution (inset ACS Paragon Plus Environment

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of Figure 3e). This result indicates that the delicate design of molecules is critical to controlling the morphologies of peptide assemblies.41-43

Figure 3. Negatively stained TEM images showing the self-assembled nanoaggregates of AMPs (0.1 mM): a) G1, b) G2, c) G3, d) R1, and e) R3. Inset of e) is a cryo-TEM image of a vesicle. 3.2. Nanostructures as targeted theranostic agents with antimicrobial activity To develop AMPs with the potential for use in therapies and diagnostics, G1 and R1 were coassembled at a molar ratio of 1:1. Their co-assembly behavior was first confirmed by 2D NMR and Fourier transform infrared (FTIR) spectroscopy (Figure 4a,b and Figures S12,13). Nuclear Overhauser effect spectroscopy (NOESY) of the G1 and R1 mixture (0.5 mM), hereafter referred to as G1R1, showed intermolecular contacts between the alpha proton (αH) of histidine in G1 and the benzyl proton of tyrosine in R1, indicating that the nanostructures were composed of the co-assembled functional AMPs. The FTIR spectra showed that a strong tendency to form β-turn structure of G1 is reduced after the addition of R1 (Figure 4b). The characteristic amide I and II bands of the β-turn structure appeared at 1674 cm-1 and 1541 cm-1, respectively.44 Subsequently, we investigated the Gd3+-binding affinity of co-AMPs by monitoring the C-N and N-C-N stretching vibrations (vC-N and vN-C-N) in the imidazole side ACS Paragon Plus Environment

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chain of histidine (Figure 4c and Figure S14).44-46 After the addition of Gd3+, the vC-N at 1138 cm-1 was shifted to 1153 cm-1 and vN-C-N at 1183 cm-1 disappeared, suggesting chelation of the histidine residue of GHK in

Figure 4. a) NOESY of co-assembled G1 and R1 (for G1R1) with a 1:1 molar ratio (600 MHz, tm = 500 ms, n = 128; tm = mixing time, n = number of scans). FTIR spectra of b) G1, R1, and G1R1 and c) after addition of Gd3+ to G1R1. TEM images of co-AMPs after complexation with Gd3+ in 0.1 mM solution: d) G1R1, e) G2R1, f) G3R1, g) G1R3, h) G2R3, and i) G3R3. f) and i) were obtained by cryo-TEM experiments. Inset of h) is a fluorescence microscopy image. ACS Paragon Plus Environment

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G1 with Gd3+ (Figure S6).31 The association between G1 and Gd3+ was further confirmed by MALDITOF/TOF-MS (Figure S15).47 Signals were observed at 973 m/z, which corresponded to [G1 + Gd + K + H2O]+. Gd3+-complexed co-assembled peptide nanostructures were confirmed by TEM (Figure 4d-i). A TEM image of Gd3+-complexed G1R1 (Gd3+-G1R1) showed micellar structures with a remarkable enhancement of mass contrast without staining (Figure 4d). The Gd3+-complexed, co-assembled G2 and R1 (Gd3+-G2R1) with enhanced hydrophobicity formed nanofibers with a diameter of ~7 nm (Figure 4e). A cryo-TEM image of the mixture of G3 with R1 (Gd3+-G3R1) revealed the formation of vesicular structures (Figure 4f). The strong tendency of R1 to assemble in ribbons was sterically hindered by neighboring peptides induced by complexation of Gd3+ with GHK-attached AMPs during aqueous selfassembly. G1 mixed with R3 (Gd3+-G1R3) also resulted in the formation of vesicles (Figure 4g). However, interestingly, a TEM image of co-assembled G2 and G3 with R3 (Gd3+-G2R3 and Gd3+G3R3, respectively) displayed a sheet-like structure (Figure 4h,i). A TEM image of Gd3+-G2R3 showed crumpled sheets of several microns or more (Figure 4h). The presence of a sheet-like structure in aqueous solution was further confirmed by fluorescence optical microscopy (inset of Figure 4h). The molecular organization for the formation of 2D structures was investigated by atomic force microscopy (AFM, Figure S16). From the AFM images it is clear that Gd3+-G2R3 formed a planar structure with a thickness of ~5.2 nm, confirmed by height profile (inset of Figure S16a) which indicates that Gd3+G2R3 packed in a bilayer arrangement.48 More interestingly, Gd3+-G3R3 showed the formation of 2D porous sheets that were a few micrometers in size. Although a planar network can be induced by increasing the intermolecular interactions of G3 and R3 with a longer backbone structure, a specific mechanism is being investigated to vary the molecular structure. Even though sheet-like structures formed from the self-assembly of peptides have been reported,49-51 as far as we know, this is the first report on the formation of 2D porous network structure in aqueous solution. The aqueous stability of an interconnected network structure could be explained by the effects of cross-linking of neighboring peptides by Gd3+-chelation.27 ACS Paragon Plus Environment

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Figure 5. a) Antibacterial activities of Gd3+-chelated co-AMPs (25 µM) toward Gram-negative E. coli ATCC8739 and Gram-positive S. aureus ATCC6538p evaluated by LB liquid medium turbidity assays. b) Optical density at 600 nm of an untreated or Gd3+-G3R3-treated E. coli ATCC8739 suspension as a function of time and concentration of Gd3+-G3R3. The antimicrobial activities of Gd3+-chelated co-AMPs were tested against a cell suspension of Gram-negative E. coli and Gram-positive S. aureus using an LB liquid medium turbidity assay.52 We cultured E. coli and S. aureus in LB liquid medium for 12 h, until the mixture become turbid, suggesting the proliferation of bacteria. The amount of growth inhibition of Gd3+-chelated co-AMP aggregates was calculated by subtracting the percentage turbidity of each treatment from that of the control, which was considered 100% (25 µM, Figure 5a). All Gd3+-chelated co-AMP aggregates were found to be active against E. coli and S. aureus. However, the growth of Gram-negative E. coli was inhibited more by Gd3+-chelated co-AMPs compared to that of Gram-positive S. aureus. This may be attributed to the thicker cell walls of Gram-positive bacteria, which may affect the access of Gd3+-chelated co-AMPs to bacteria. In addition, nanoaggregates with antibacterial activity formed from Gd3+-chelated co-AMPs of G1, G2, and G3 with R1 indicated that, first, introducing hydrophobic and positive charge units into peptide helped determine antimicrobial activity; second, peptide length is also important to antimicrobial activity. As a result, Gd3+-G3R1 showed stronger antibacterial activity than that of Gd3+G1R1 and Gd3+-G2R1. Gd3+-G1R3 also prevented bacterial growth. However, Gd3+-G2R3 and Gd3+G3R3, which had sheet-like structures, showed lesser antibacterial activity than that of Gd3+-G1R3 with a vesicular structure. In fact, the porous sheets of Gd3+-G3R3 showed no antibacterial effect at up to 2 h ACS Paragon Plus Environment

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of incubation, but did show antibacterial activity after 12 h (Figure 5b). This tendency was even more apparent when the concentration was increased. Sheets with relatively larger aggregates had difficulty permeating bacterial membranes and required time for penetration.25 For practical use of co-AMPs as antimicrobial agents, the hemolysis assay was carried out in mRBCs used as a representative mammalian cell.30 As shown in Figure S17, both Gd3+-G1R3 and Gd3+-G3R3 showed low hemolysis at a concentration of 25 µM where antibacterial behavior was observed. The measured values of 15.2% for Gd3+-G1R3 and 10.9% for Gd3+-G3R3 are not considered to be significant for in vitro analysis.53 The hemolytic activity of Gd3+-G1R3 and Gd3+-G3R3 against mRBCs increased with increasing concentration of co-AMPs. These results suggest that the developed co-AMPs possess antimicrobial potency without significant hemolytic activity to mammalian cells. The cytotoxic activity of Gd3+-chelated co-AMPs was evaluated against MCF-10A (human epithelial cell line), NIH3T3 (mouse embryo fibroblast cell line) and HeLa (human tumor cell line) cells to confirm their biocompatibility in theranostic applications (Figures S18, S19 and Figure 6a). Cell viability was clearly reduced only in HeLa cells when using a 25 µM AMP-solution above the CMC. The designed AMPs, which contained cationic and hydrophobic amino acids, could disrupt the membranes of cancer cells by electrostatic and hydrophobic interactions with cell membrane and thus led to decreased cell viability. However, Gd3+-G2R3 and Gd3+-G3R3 with sheet-like structures showed relatively low cytotoxicity in HeLa cells. Gd3+-G2R3 and Gd3+-G3R3 resulted in cell viabilities of 97% and 90%, respectively (Figure 6a). Sheets of a few micrometers or more seemed to have difficulty penetrating the HeLa cells membrane which corresponds to the antibacterial activity study results. In fact, the cRGD peptide recognizes HeLa cells with αvβ3 integrin receptors and induces receptormediated endocytosis for cellular uptake,27 enhancing the intracellular drug delivery efficiency. The encapsulation ability of co-AMPs as carriers was investigated by fluorescence spectroscopy using Nile Red as a hydrophobic drug model (Figure S20). The characteristic emission peak of water-insoluble Nile Red was observed in all solutions with Gd3+-chelated co-AMPs (25 µM, λex = 550 nm), confirming the successful loading of Nile ACS Paragon Plus Environment

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Figure 6. a) Cytotoxicity in HeLa cells of various Gd3+-chelated co-AMPs at different concentrations (25–50 µM) determined by CCK-8 assay after 12 h of treatment. b) Flow cytometry histogram of HeLa cells incubated with Nile Red-loaded Gd3+-chelated co-AMP nanostructures for 1 h, and (inset) MFI of Nile Red in HeLa cells. c) CLSM images of HeLa and NIH3T3 cells treated with Nile Red-loaded porous networks of Gd3+-G3R3 (1 h after incubation): left, Nile Red (red); middle, nuclei stained with DAPI (blue); right, merged images. d) Quantitative analysis of the uptake of Nile Red-loaded Gd3+G3R3 by HeLa cells represented with MFI. e) T1 relaxivity plots of Gd3+-chelating nanostructures of G1R3, G2R3, and G3R3 (0.2 mM), as a function of Gd3+-concentration ([Gd3+]) (4.7 T, 25 °C) and (inset) T1-weighted MR images of Gd3+-G3R3. ACS Paragon Plus Environment

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Red in the nanoaggregates. It is noted that DOX, as a clinical anticancer drug, was also successfully encapsulated into co-AMPs (Figure S21). The cellular uptake and intracellular distribution of Nile Red-loaded, Gd3+-chelated co-AMPs were examined in HeLa and NIH3T3 cells (Figure 6b-d and Figure S22).54 Rapid αvβ3-mediated endocytosis facilitated the internalization of cRGD-encoded nanostructures into HeLa cells. CLSM showed that, once in cells, the majority of Nile Red-loaded nanostructures were localized in cytoplasmic compartments (Figure 6c and Figure S22). Intracellular delivery was quantitatively evaluated using fluorescence-activated cell sorting analysis (FACS, Figure 6b). The internalization of Gd3+-chelated coAMPs increased as the number of hydrophobic Py amino acid residues and the repeating Py-Arg units of AMPs increased (see results of Gd3+-G1R1, G2R1, G3R1, and G1R3). Noticeably, MFI of Gd3+G2R3 was ~4.2-fold lower than that of Gd3+-G3R3 (inset of Figure 6b), despite both having sheet-like structures (Figure 4h,i). As mentioned, the size of the Gd3+-G2R3 sheet was significantly larger than that of Gd3+-G3R3. This can be explained by the fact that larger-size of 2D sheets are generally not beneficial for drug delivery, but they can load a larger amount of a drug; thus, these sheets are a suitable drug delivery vehicle as long as the sheet with appropriate size can be introduced into the cells. Specific tumor cell-targeted intracellular delivery of Gd3+-G3R3 containing cRGD was examined by employing normal NIH3T3 cells with low αvβ3 integrin expression as a negative control.55 No noticeable toxicity was detected when Gd3+-chelated co-AMPs were incubated with NIH3T3 cells for 12 h (Figure S19). The cellular uptake of Nile Red-loaded Gd3+-G3R3 was assessed in both HeLa and NIH3T3 cells (Figure 6c,d). In contrast to the result obtained with HeLa cells, no significant emission of Nile Red was observed in NIH3T3 cells demonstrating the tumor-targeting drug delivery ability of co-AMPs. The MFIs obtained by FACS showed quantitative differences (Figure 6d). To further examine the potential diagnostic application of co-AMPs nanostructures, the ability of Gd3+-chelated co-AMPs to serve as T1 MRI agents was investigated (Figure 6e and Figure S23). The proton relaxivity (r1) of a T1 agent can be determined by paramagnetic enhancement of the longitudinal ACS Paragon Plus Environment

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water proton relaxation rate (R1 = 1/T1, s-1) per Gd3+ concentration (mM). The amount of Gd3+ chelated by co-AMPs containing GHK was measured by ICP-MS. We found that the r1 value of Gd3+-G3R3, 3.6 mM-1S-1, was the highest among the Gd3+-chelated co-AMP contrast agents and showed the strongest MR signal (Figure 6e). This could be explained by increased molecular weight induced by large, selfassembled 2D planar sheet, which slowed the correlation time of Gd3+-G3R3 compared to that of other nanostructured agents.56 The rotational modes of Gd3+-chelates can be restricted by the assembled nanostructures, increasing the r1 and MRI contrast. These results agreed with the fact that the longitudinal relaxivities of Gd3+-chelates are dependent on molecular weight: the higher molecular weight, the higher the longitudinal relaxivities. The calculated r1 value of Gd3+-G3R3 was similar to that of a commercial single molecular Gd3+-DOTA contrast agent of 1 M (~4 mM-1S-1). However, because we used the diluted Gd3+-G3R3 solution (0.2 mM) at a 1:1 molar ratio with G3 and R3, this supramolecular approach using GHK moieties to develop a T1 contrast MRI agent allows the opportunity to improve the performance of the agent.

4. Conclusions We developed supramolecular theranostic nanoagents with antimicrobial activity based on the selfassembly of AMPs capable of Gd3+ conjugation and tumor-targeting in aqueous solution. The relationship between the morphology of nanostructures and their characteristics including antibacterial effect, MRI efficacy, hydrophobic drug loading and delivery capability was investigated using various nanostructures such as spheres, cylinders, vesicles, and sheet-like structures. Interestingly, 2D planar sheets with and without pores showed antibacterial activity against Gram-negative E. coli and Grampositive S. aureus. However, lesser antibacterial effect than that of micelles was observed. We also found that the developed AMP nanoagents showed anticancer activity, but that the large sheet-like structures showed relatively poor activity. These low antibacterial and anticancer activities could be explained by the inability of sheets of several micrometers or more to penetrate and disrupt cell membranes. In contrast, in terms of drug-loading capacity and specific cell-targeting ability, 2D sheets ACS Paragon Plus Environment

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showed better performance than other nanoagents because of the larger hydrophobic capacity and receptor-binding areas. Furthermore, restriction of the rotational modes of Gd3+-chelates by supramolecular 2D sheets assembled from single peptides resulted in increased T1 relaxivity for contrast-enhanced MRI. Therefore, we anticipate that the performance of theranostic nanostructured agents can be tuned by rational design of their chemical structures and precise control of their aqueous assembly. We expect that results of this study may facilitate the development of a new class of theranostic agents against drug-resistant bacteria.

ASSOCIATED CONTENT Supporting Information Synthesis and identification of a series of AMPs, co-assembly behavior of AMPs in nanostructures, and the target-selective intracellular delivery of these AMPs. This material is available free of charge via internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] *E-mail: [email protected]

Notes The authors declare no competing financial interest.

Acknowledgments This research was supported by Basic Science Research Program (2016R1A2B4012322) through the National Research Foundation of Korea (NRF), Chungnam National University Research Fund, the ACS Paragon Plus Environment

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Korea Basic Science Institute grant (D36402) and R&D Convergence Program (CAP-16-03-KRIBB) of NST (National Reserach Council of Science & Technology). We acknowledge Bo Kyoung Choi and Yoon Seok Lee for their contribution to evaluation of cell viability and antimicrobial activity and HaeKap Cheong and Eun-Hee Kim for 1H NMR measurement.

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Structure-Dependent Antimicrobial Theranostic Functions of Self-Assembled Short-Peptide Nanoagents

Inhye Kim, Seon-Mi Jin, Eun Hee Han, Eunhee Ko, MiJa Ahn, Woo-Young Bang, Jeong-Kyu Bang,* Eunji Lee*

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