Enzymatic Regulation of Self-Assembling Peptide A9K2

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Enzymatic Regulation of Self-Assembling Peptide AK Nanostructures and Hydrogelation with Highly Selective Antibacterial Activities Jingkun Bai, Cuixia Chen, Jingxin Wang, Yu Zhang, Henry Cox, Jing Zhang, Yuming Wang, Jeffrey Penny, Thomas Andrew Waigh, Jian Ren Lu, and Hai Xu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b03770 • Publication Date (Web): 31 May 2016 Downloaded from http://pubs.acs.org on June 2, 2016

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ACS Applied Materials & Interfaces

Enzymatic Regulation of Self-Assembling Peptide A9K2 Nanostructures and Hydrogelation with Highly Selective Antibacterial Activities Jingkun Bai,†,# Cuixia Chen,†,# Jingxin Wang,† Yu Zhang,† Henry Cox, Jing Zhang,‡ Yuming Wang,† Jeffrey Penny,‡ Thomas Waigh, ‡ Jian R. Lu*,‡ and Hai Xu*,†

†

State Key Laboratory of Heavy Oil Processing and Centre for Bioengineering and

Biotechnology, China University of Petroleum (East China), 66 Changjiang West Road, Qingdao 266580, China. ‡

Biological Physics Laboratory, School of Physics and Astronomy and Manchester

Pharmacy School, University of Manchester, Schuster Building, Manchester M13 9PL, UK #

Contributed equally to this work

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ACS Paragon Plus Environment


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ABSTRACT Hydrogels offer great potential for many biomedical and technological applications. For clinical uses, hydrogels that act as scaffold materials for cell culture, regenerative medicine, and drug delivery are required to have bactericidal properties. The amphiphilic peptide A9K2 was designed to effectively inhibit bacterial growth via a mechanism of membrane permeabilization. The present study demonstrated that addition of fetal bovine serum (FBS) or plasma amine oxidase (PAO) induced a sol-gel transition in A9K2 aqueous solutions. The transformation of A9K2 molecules catalyzed by lysyl oxidase (LO) in FBS or PAO accounted for the hydrogelation. Importantly, the enzymatic A9K2 hydrogel displayed high antibacterial ability against both Gram-negative (G-) and Gram-positive (G+) bacterial strains whilst showing extremely low mammalian cell cytotoxicity, thus demonstrating good biocompatibility. Under established co-culture conditions, the peptide hydrogel showed excellent selectivity by favoring the adherence and spreading of mammalian cells, while killing pathogenic bacteria, thus avoiding bacterial contamination. These advantages endow the enzymatic A9K2 hydrogel with great potential for biomedical applications.

KEYWORDS: short peptide, self-assembly, enzyme, hydrogel, antibacterial activity, cell selectivity

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1. INTRODUCTION Hydrogels are attractive for numerous potential applications in regenerative medicine, cell culturing, and tissue engineering, in which they act as scaffolds to either engineer new tissues or encapsulate payloads (cells or drugs) and deliver them to the desired sites.1-3 To function appropriately in these applications, hydrogels should meet an array of criteria, including several critical physical and biological parameters, such as appropriate elasticity,

porosity,

cell-based

degradability,

cell

adhesion,

cell

proliferation,

biocompatibility and a lack of appreciable cytotoxicity. Due to their biological origins and ease of gelation (often possible under physiological conditions and in the absence of toxic cross-linkers), self-assembling hydrogels formed from short designed peptides are particularly attractive.4-7 Aside from showing low cytotoxicity and adequate biocompatibility, peptide hydrogels consist of three-dimensional (3D) networks of self-assembled peptide nanofibers and contain over 99% water, which is structurally and compositionally similar to the natural extracellular matrix (ECM). Furthermore, peptide self-assembly is often responsive to environmental stimuli, so the hydrogels formed can provide dynamic microenvironments for cell-matrix and cell-cell interactions upon physiochemical and biological cues. The potential for bacterial growth on hydrogels should be carefully considered in their clinical applications. Bacterial growth that drives infections in cell culture, tissue engineering, and implant surgery are common, with serious health implications for patients.8-11 The sources of bacterial contaminations are numerous and can be subtle, 3



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often arising from contaminated culture media, glassware and devices, airborne microbes in the operating theatres, host contamination or haematogenous seeding, and even skin and nasal shedding from a member of the operative team.8-12 Furthermore, the risk of contamination could be higher with multifilament and woven sutures or porous materials than that with monofilament sutures or solid materials.9, 11, 13 Therefore, it is desirable to create hydrogels with intrinsic antibacterial activity for biomedical applications. Such hydrogels could avoid the need for harsh sterilization procedures that may change the material’s performance, and mitigate excessive use of conventional antibiotics that readily leads to antibacterial resistance. A further confounding factor is that implant infections can be extremely resistant to conventional antibiotics and host defenses.10 If hydrogels can kill bacteria via alternative means to conventional antibiotics, they will present a significant clinical opportunity. As one of the most promising candidates for future antibiotic drugs, antibacterial peptides (AMPs) kill pathogenic bacteria via physical permeation and disruption of cell membranes, rather than interacting with specific receptors.14-17 Such a mode of action can help to disrupt the development of bacterial resistance.14-17 AMPs are often disordered in water, and most are unable to self-assemble into well-defined architectures in aqueous solution to create supramolecular hydrogels, although they are thought to adopt amphipathic, well-ordered secondary structures (e.g., α-helices and β-sheets) when they interact with plasma membranes. However, certain synthetic AMPs that emulate fundamental features of natural AMPs (e.g. net positive charges and amphipathicity), and 4


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structurally resemble traditional surfactants, can self-assemble in aqueous solution to form well-defined nanostructures.18-22 Lipopeptides (e.g. C16-KKK) and surfactant-like peptides (e.g. A9K and A9K2) provide typical examples.20-22 In their self-assembly, the hydrophobic interaction between hydrophobic tails (aliphatic chains and consecutive hydrophobic amino acid residues) seems to play a more important role than the electrostatic interaction between hydrophilic heads and the hydrogen bonding between peptide backbones. Therefore, the peptide aggregates (e.g. micelles and short nanorods) are usually small in size and unbranched, similar to those of surfactants.20-22 As there is little cross-linking and entanglement among these small aggregates, the self-assembly of the AMPs is still unable to create supramolecular hydrogels. A9K2 is a surfactant-like peptide, designed by us, which possess two charged lysine residues as the hydrophilic head.22 The peptide can form micelles and short nanorods in aqueous solution via self-assembly and kill bacteria via a mechanism of membrane permeabilization. As an important biological stimulus, enzymes have been exploited to manipulate the peptide’s self-assembly and hydrogelation.23-25 The enzymes can convert non-assembling precursors into self-assembling building blocks or induce cross-linking of existing aggregates, eventually producing fibrous hydrogels. Lysyl oxidase (LO) plays an important role in the formation and repairing of the natural ECM, via oxidation of lysine or hydroxylysine residues of major ECM proteins such as collagen and elastin, and its action can lead to cross-linking.26

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Therefore, we here investigated the self-assembly and hydrogelation of A9K2 in the presence of LO and plasma amine oxidase (PAO), as well as the mechanisms involved, by the combined use of transmission electron microscopy (TEM), atomic force microscopy (AFM), circular dichroism (CD) spectroscopy, rheology, matrix assisted laser desorption/ionization time of flight mass spectroscopy (MALDI-TOF MS), and reversed-phase high performance liquid chromatography (RP-HPLC). To establish relevance for practical applications, we then examined whether the enzymatic A9K2 hydrogels inhibited bacterial growth. Such hydrogels have clearly showed effective bactericidal effects while displaying low cytotoxicity against mammalian cells and good biocompatibility. We demonstrated these performances by measuring bacterial viability and lactate dehydrogenase (LDH) release, and by employing enzyme-linked immunosorbent assay (ELISA) to quantify proinflammatory cytokine production. To further test the selectivity of the hydrogels, a co-culture system containing mammalian cells and pathogenic bacteria was created. Enzyme-mediated peptide self-assembly and hydrogelation are thought to be of great importance for biomedical applications, because of the critical roles of enzymes in almost all metabolic processes, as well as their high substrate specificity and overexpression in some injuries (e.g. bacterial infections, cancers, and bleeding).

2. MATERIALS AND METHODS 2.1. Materials

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Unless specifically noted, all materials were used as received. The materials used for peptide synthesis such as Fmoc-protected amino acids, coupling reagents, resin, and solvents were purchased from GL Biochem Ltd. (Shanghai, China) and Bo Maijie Technology (Beijing, China). The solvents including piperidine, dichloromethane and N,N'-dimethyl formamide were redistilled before use. Calcein-AM and propidium iodide (PI) were obtained from KeyGen Biotechnology Co. (Nanjing, China) and SYTO 9 was bought from Life Technologies (Carlsbad, USA). Other chemical reagents were purchased from Sigma (St. Louis, MO, USA). Water used in all experiments was processed from a Milli-Q Biocel ultrapure water system (Millipore), with a minimum resistivity of 18.2 MΩ·cm. The following bacteria were used: Gram-negative (G-) strains Escherichia coli DH5a (E. coli DH5a) and Pseudomonas aeruginosa（P. aeruginosa）and Gram-positive (G+) strains Bacillus subtilis 168 (B. subtilis 168) and Staphylococcus aureus (S. aureus). All the bacterial strains were supplied by the China Centre of Industrial Culture Collection. E. coli DH5a and P. aeruginosa were incubated with LB medium (tryptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L, pH of 7.0) while B. subtilis 168 and S. aureus were cultured in beef medium (glucose 60 g/L, beef extract 10 g/L, peptone 10 g/L, yeast extract 10 g/L, and NaCl 5 g/L at pH 7.0).22 NIH Swiss mouse embryo fibroblast NIH 3T3 cells and human embryonic kidney 293 (HEK293) cells were purchased from the Shanghai Institute for Biological Science, CAS, cultured in DMEM (Dulbecco’s modiﬁed Eagle’s medium, pH 7.4) with 10% (v/v) FBS (fetal bovine serum) at 37 oC under 5% CO2.22 7



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2.2. Peptide Synthesis and Hydrogel Preparation Peptide synthesis was performed on a commercial CEM Liberty microwave peptide synthesizer, based on Fmoc solid-phase chemistry. All the peptides were synthesized via coupling reaction from their C-termini to N-termini. To make their C-termini amidated, Rink amide MBHA resin was used, and their N-termini were capped with acetic anhydride before cleavage from the resin. A detailed description of the peptide synthesis and subsequent purification procedures has been given in our previous works.19, 21, 27 The final product purity (>98%) was confirmed with MALDI-TOF MS and RP-HPLC analysis. The synthesized A9K2 was directly dissolved in Hepes buffer (25 mM, pH 7.4) to get a stock solution of 8 mM (7.64 mg/mL or 0.764% (w/v)), which was incubated for 1 day at room temperature prior to use. The stock solution was a clear and freely flowing solution. To mediate gelation, different methods were then applied, including: (1) the addition of FBS at concentrations of 1%, 2%, and 4% (v/v); (2) the addition of PAO at concentrations of 5, 10, and 20 U/mL and 10 µM of Cu2+; (3) the addition of NaCl at concentrations of 10, 50, and 100 mM. The changes in peptide concentration and pH were negligible after these treatments. Finally, the resulting peptide solutions were incubated at 37 oC for the indicated time periods. 2.3. AFM and TEM Before starting AFM imaging, surface immobilized peptide samples must be carefully prepared. Briefly, a small amount (~10 µL) of peptide solution or hydrogel was deposited 8


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on a freshly cleaved mica surface. After adsorption for 10 s, the substrate surface was gently rinsed with Milli-Q water and then dried by purging with N2. When all traces of water had disappeared (usually within 2 min), AFM height images were acquired on a Veeco Nanoscope IVa MultiMode AFM microscope (Digital Instruments, Santa Barbara, CA), in tapping mode by using TESP silicon probes. TEM measurements were performed at an accelerating voltage of 200 kV on a JEOL JEM-2100 UHR instrument. Before starting TEM imaging, a drop of peptide solution or hydrogel was put on a glass slide and a 300-mesh copper grid was then placed on the top of the liquid drop. After short contact, the grid was negatively stained with uranyl acetate (2%, w/v) for approximately 3 min, followed by removal of any excess solution with filter paper. Finally, the grid was air-dried and viewed on the TEM instrument. 2.4. Rheology The rheological properties of A9K2 hydrogels were recorded at 25 oC on a Thermo Scientific Haake MARS III modular rheometer. The cone-plate geometry was as follows: cone angle, 2.0°; diameter, 34.995 mm; truncation, 0.105 mm. During measurements, a Peltier temperature module was utilized to control the temperature and a solvent trap to cover the cone-plate cell to prevent water evaporation. First of all, strain sweeps (from 0.01% to 100% strain, 1 Hz (6.28 rad/s)) were carried out to fix linear viscoelastic regime,27 in which subsequent dynamic frequency sweeps (0.01-10 Hz and 1% strain) as well as shear-thinning and recovery experiments were performed. In shear-thinning and recovery experiments, after a dynamic time sweep (1 Hz, 1% strain, from 0 to 30 min), 9



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1000% strain was applied for 3 min to shear-thin the gel, followed by an immediate strain decrease to 1% and another dynamic time sweep (1 Hz, 1% strain, 30 min duration). Such a shear-thin and recovery cycle was conducted twice. 2.5. CD Spectroscopy CD measurements were performed at room temperature on a MOS-450/AF-CD spectrophotometer (Biologic, France). The CD spectra were taken in a 0.1 mm quartz cell, by wavelength scanning from 260 to 190 nm with a 0.5 nm step. The resulting CD spectra were the average of six independent measurements for each sample, presented as [θ] (deg·cm2·dmol-1) versus wavelength (nm). 2.6. MALDI-TOF MS and RP-HPLC Mass spectrometry measurements were performed on a Bruker Microflex MALDI-TOF mass spectrometer. Positive-ion mode spectra were recorded with m/z ranging from 400 to 3150 at a laser frequency of 60 Hz. Other instrumental parameters were as follows: ion source I voltage, 19.0 kV; ion source II voltage, 15.7 kV; lens voltage, 9.2 kV; accelerating voltage, 19.0 kV; reflection voltage, 20.0 kV; extraction delay time, 140 ns. The matrix used in this study was 4-hydroxy-α-cyanocinnamic acid (HCCA), which was dissolved in 30% (v/v) acetonitrile containing 0.1% (v/v) trifluoroacetic acid (TFA) to obtain a saturated matrix solution. For the sample preparation, 1 µL of peptide solution was mixed with 1 µL of the saturated HCCA solution. Before such a mixing, the 8 mM A9K2 solution and the enzymatic peptide hydrogel were subjected to a 10-fold dilution using 25 mM Hepes (pH 7.4). Then, 1 µL of the mixture was dropped on a polished steel 10


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sample target and air-dried, immediately followed by MS measurements. Each of the spectra collected was the integration of 100 individual measurements. HPLC profiles of A9K2 before and after addition of PAO were recorded on a Waters 2695 Alliance HPLC system equipped with a C18 reversed-phase column (4.6 mm × 150 mm). The eluting system consisted of eluent A (0.1% (v/v) TFA in water) and eluent B (0.1% (v/v) TFA in acetonitrile). After being loaded onto the C18 column, samples were first analyzed using an isocratic elution consisting of 95% eluent A and 5% eluent B for 1 min, followed by a linear gradient elution to 5% eluent A and 95% eluent B for 39 min. Finally, a linear gradient elution to 95% eluent A and 5% eluent B for 5 min was applied to reset the system. Other instrumental conditions were as follows: eluent flow rate of 0.6 mL/min and UV detection at 214 nm. The 8 mM A9K2 solution and the enzymatic hydrogel were subjected to an 8-fold dilution in 25 mM Hepes (pH 7.4) for such an analysis. 2.7. Bacterial Viability Assays A9K2 hydrogels used in the assessment of bacterial viability were prepared in a 96-well tissue culture polystyrene (TCPS) plate and an 8-well borosilicate glass plate. Specifically, aliquots (96 µL) of the A9K2 stock solution were placed in each wells, followed by addition of 4 µL FBS. After incubation at 37 oC for two weeks in a CO2 incubator, 100 µL of LB or beef medium was introduced into the wells containing the newly forming peptide gel. Samples were subject to equilibration overnight at 37 oC.

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After removing the medium used for equilibration, aliquots (100 µL) of bacterial suspensions (1×106 CFU/mL) were added onto the hydrogel surface. Following incubation for 24 h at 37 oC, 100 µL of a Live/Dead fluorochrome solution, containing 15 µM SYTO 9 and 30 µM PI, was introduced into each well of the 8-well plate. After staining for 30 min at ambient temperature, bacteria were directly viewed using a Nikon A1-si confocal laser scanning microscope (Japan) at a 40× magnification. When excited at 488 nm with an Ar/Kr laser, viable bacteria with intact plasma membranes exhibited green fluorescence while dead bacteria with disrupted membranes fluoresced red.28-31 In order to clearly observe the bacteria in the solution above the hydrogels, the solution was carefully removed from the top of the hydrogels after Live/Dead staining and then subjected to centrifugation for 5 min at 8000 rpm. After washing three times with PBS, the collected bacteria were re-suspended in PBS and fixed with 5% (v/v) glutaraldehyde. Finally, 5 µL of the bacterial suspension was dropped onto a freshly cleaned glass slide and observed by using an inverted fluorescence microscope (Leica DMI3000B) at a 100× magnification. Under blue exciting light, viable bacteria displayed green fluorescence, and under green exciting light, dead bacteria exhibited red fluorescence. The bacteria in the wells in the absence of peptide acted as the control. To quantitatively assess the proliferation of bacteria in the solutions above the hydrogels, the bacterial suspension was removed from the 96-well plate and transferred into individual wells of a new 96-well plate. The absorbance was read at 600 nm on a microplate reader (M2e, Molecular Device). The bacterial suspension in the wells without 12


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peptide was used as a control and the wells without bacteria served as a blank for the absorbance measurements. 2.8. LDH Assay The cytotoxicity of the enzymatic hydrogel was determined by measuring the activity of lactate dehydrogenase (LDH) released by cells. The preparation of A9K2 hydrogels in the wells of a 96-well TCPS plate was the same as above. The hydrogel formed was equilibrated overnight with DMEM. After removing the medium, NIH 3T3 mouse fibroblast cells in DMEM (1×105 cells/mL, 100 µL) were introduced onto the hydrogel surface. When the cells completely adhered to the gel surface (typically taking 6 h), the medium was removed, followed by washing the seeded cells with PBS. Then, fresh DMEM (150 µL) was added into each well. After culturing at 37 oC in a CO2 incubator for the indicated times, LDH release was determined using a commercial LDH cytotoxicity assay kit (Beyotime Institute of Biotechnology), according to the manufacturer’s instructions. Specifically, after centrifugation at 800 rpm for 5 min, 120 µL of supernatant was transferred into the wells of a new 96-well plate, followed by addition

of

60

µL

reaction

solution

containing

2-(p-iodophenyl)-3-(p-nitrophenyl)-5-tetrazolium (INT). After incubation in the dark for 30 min at room temperature, the absorbance at 490 nm (A490) was measured using a microplate reader (M2e, Molecular Device). LDH activity was calculated based on the following equation: LDH Activity = (A490/sample-A'490/blank)/(A490/max-Aʹʹ490/blank) 13



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where A490/sample and A'490/blank denote the measured absorbance values from the hydrogel-coated wells in the presence and absence of cells, respectively. A490/max denotes the absorbance from the uncoated wells that were subjected to complete membrane lysis by using a LDH releasing agent (Beyotime Institute of Biotechnology) after incubation at 37 oC, and Aʹʹ490/blank denotes the absorbance from the uncoated wells without cells. Simultaneously, the activity of LDH released from the cells cultured in the wells in the absence of peptide was determined as a control, based on the following equation: LDH Activity = (A490/control-Aʹʹ490/blank)/(A490/max-Aʹʹ490/blank) where A490/control denotes the measured absorbance value from the uncoated wells with cells. The cytotoxicity of A9K2 against a human cell line HEK293 was also assessed using the LDH assay. 2.9. Immunological Assay To test whether the peptide hydrogels induce non-specific immunological responses in a host, human lymphocytes were harvested from blood using the standard Ficoll method, and after incubation on the surface of the hydrogel for 48 h, the major cytokines TNFα and IL8 release from the lymphocytes were followed by ELISA method.22,

27, 32

Specifically, 10 mL of whole blood obtained from healthy volunteers was mixed with an equal volume of PBS, followed by addition of the diluted blood into a Ficoll solution (1.077 g/mL) to a final

1:1 ratio (v/v). Then the resulting solution was centrifuged for

15 min at 2000 rpm, and the lymphocytes were collected from a formed ring, washed 14


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with PBS three times and re-suspended in DMEM prior to use. Note that all experiments associated with human blood were carefully reviewed and approved by the Animal Experimental Ethical Committee of China University of Petroleum. A9K2 hydrogels in the wells of a 96-well TCPS plate were prepared based on the method described above. The lymphocytes in DMEM (50 µL, 2×106 cells/mL) were introduced onto the hydrogel surface and incubated for 48 h at 37 °C under 5% CO2. Then, the supernatants were removed and analyzed for the secretion of TNFα and IL8 cytokines using ELISA kits (Abcam® ab40632 and ab40687). Here, the lymphocytes activated by lipopolysaccharide (LPS) at a concentration of 2 µg/mL served as a positive control and the lymphocytes seeded on a TCPS plate without the peptide hydrogel were taken as a negative control. 2.10. Co-Culturing Assay Co-culturing experiments were performed on an 8-well TCPS plate with mammalian and bacterial cells. A9K2 hydrogels were first prepared in the wells of an 8-well plate as described above. These hydrogels were then equilibrated overnight with DMEM. After removal of the medium, NIH 3T3 cells in DMEM (1×105 cells/mL, 100 µL) were introduced onto the hydrogel surface. The cells were incubated for 4 days at 37 oC under 5% CO2, which allowed the cells to spread on the gel surface. Then, aliquots (50 µL) of bacterial suspension were introduced into the plate with a final concentration of 1×106 CFU/mL. After co-culturing for 24 h, aliquots (60 µL) of a staining solution containing SYTO 9, PI, and calcein-AM were added into the wells, followed by further incubation 15



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for 1 h at 37 oC. Cells and bacteria were directly viewed on a confocal laser scanning microscope (Nikon A1-si). When excited at 488 nm with an Ar/Kr laser, both viable bacteria and NIH 3T3 cells exhibited green fluorescence while dead ones exhibited red fluorescence. The wells without the peptide gel acted as a control.

3. RESULTS AND DISCUSSION 3.1. Enzymatic Hydrogelation of A9K2

Figure 1. Optical images of the aqueous solution of A9K2 in presence of a) FBS (1%, 2%, and 4%) and b) PAO (5, 10, and 20 U/mL). The peptide solution (8 mM or 7.64 mg/mL) was a clear and free flowing sol, irrespective of incubation time (as a control). After incubation with 2% and 4% PBS or 10 and 20 U/mL PAO for 2 weeks, the peptide solution formed clear and self-supporting hydrogels. The peptide solution was aged for 24 h at room temperature before addition of FBS or PAO.

Our previous study has shown that A9K2 self-assembled into micelles and short nanorods, and demonstrated an effective bactericidal effect at concentrations as low as 16


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0.1 mg/mL.22 Because lysine residues are distributed on the assemblies’ surfaces, they can act as the substrates for LO and PAO. With respect to multidomain peptides (MDPs) containing lysine residues, Bakota et al have demonstrated that the presence of FBS or addition of PAO brought about the enzymatic cross-linking of their self-assembled nanofibers, thus enhancing the mechanical strength of the nanofibrous hydrogel formed.33 Here, the A9K2 solution of 8 mM, or 7.64 mg/mL, was a clear and free flowing solution, irrespective of incubation time (the control in Figure 1). Addition of FBS or PAO at the indicated concentrations caused the peptide solution to form hydrogels after incubation for 2 weeks, as shown in Figure 1. The oxidation of lysine residues to allysines (α-aminoadipic δ-semialdehyde) catalyzed by LO in FBS or PAO, and the subsequent non-enzymatic condensation reactions could be responsible for the sol-gel transition. Although LO plays an important role in the formation and repairing of the natural ECM by oxidizing lysine or hydroxylysine residues of fibrous proteins such as collagen and elastin leading to their covalent cross-linking,26 it is not commercially available. Because PAO has a similar mechanism of action to LO and is commercially available,33-35 PAO was used for the following mechanistic studies. To confirm the above hypothesis, we denatured PAO by incubation at 100 oC for 10 h, and the denatured enzyme was then added into the A9K2 solution. Furthermore, because 2-butynylamine is an irreversible inactivator of PAO,36 it was also introduced into the peptide solution at a concentration of 50 mM, followed by addition of PAO. In spite of other conditions remaining the same as those that induced gelation (20 U/mL PAO), no 17



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gelation was observed in the two cases, as shown in Figure S1. Importantly, for the peptide solutions which only contained native PAO, MALDI-TOF MS spectra indicated the formation of inter- and intra-molecular lysinorleucines (Figure 2). These lysinorleucines are the products of sequential condensation reactions, which include the Schiff base formation between the enzymatically formed allysine and the ε-amine of lysine and the aldol condensation of allysines, further condensation of the aldol condensation product and dehydrolysinonorleucine as well as the simultaneous reduction of dehydrolysinonorleucine to lysinonorleucin (Figure S2).37 These results suggested that the observed sol-gel transition of A9K2 is enzyme-triggered.

Figure 2. a) MALDI TOF mass spectra of A9K2 before and after addition of PAO, indicating the formation of intra-molecular and inter-molecular lysinorleucines after addition of PAO. b) Molecular 18


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structure of intra-molecular lysinorleucine of A9K2 that has a theoretical molecular weight (MW) of 938.1. The molecule showed a measured MS peak at m/z of 961.0, which can be ascribed to its Na+ cationization. c) Molecular structure of inter-molecular lysinorleucine A9K2 that has a theoretical MW of 1893.2. The molecule showed a measured MS peak at m/z of 1916.1, corresponding to its Na+ cationization.

Figure 3. a) Rheological frequency sweeps at 1% strain from 0.01 to 10 Hz for the solution of A9K2 before and after addition of 20 U/mL PAO. The A9K2 solution of 8 mM (7.64 mg/mL) was aged for 24 h at room temperature before the enzyme addition, and the enzymatic incubation was performed at 37 o

C. b) Gel shear-thinning and recovery. After loading the peptide gel that was obtained following 19



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two-week incubation at 37 oC after addition of 20 U/mL PAO, a dynamic time sweeping (1 Hz and 1% strain) was undertaken for 30 min. Then, 1000% strain was applied for 3 min, followed by an immediate strain reduction to 1% and another dynamic time sweeping (also 30 min duration). Such a cycle was performed twice. All rheological measurements were performed at 25 oC.

Rheological measurements also indicated the enzyme-triggered sol-gel transition of A9K2. As shown in Figure 3a, after addition of PAO and incubation for two weeks, the gel showed storage moduli (Gʹ) of 300-400 Pa, both Gʹ and the loss moduli (Gʹʹ) remained nearly constant over the measured frequency range (0.01-10 Hz, 1% strain), and Gʹ was always an order of magnitude larger than Gʹʹ. These results suggested the formation of a rigid solid-like hydrogel with considerable cross-linking. Furthermore, upon application of 1000% strain, the gel immediately turned into a liquid, with its Gʹ being around 1-2 Pa and less than Gʹʹ, and upon cessation of shear, the gel recovered instantly (Figure 3b). The properties of rapid shear-thinning and immediate recovery suggested that the gel is most likely to be a physically cross-linked gel, since the physical connections can easily reform.27 Additionally, increasing the incubation time from 2 to 15 weeks induced little variation in the rheological properties (Figure 3a). As shown in Figure 4a, short nanorods and micelles were formed in the A9K2 solution of 8 mM (7.64 mg/mL) after incubation for 1 day, and there was little variation in self-assembled nanostructures with further increasing incubation time. Because the sizes of these nanostructures were relatively small (e.g. the diameter and length of the

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nanorods being ~4.5 nm and 100 nm, respectively, Figure S3) and there was little entanglement among them, the peptide solution was always a free flowing sol, irrespective of incubation time. Accompanying the enzyme-triggered hydrogelation, we observed the formation of considerable longer nanofibers, which showed increased entanglement and significant cross-linking (Figure 4b and 4c). Furthermore, there was little variation in the heights of self-assembled nanostructures during this morphological transition, i.e. both nanorods and nanofibers had similar heights of some 4.5 nm (Figure S3). In addition, β-sheet secondary structures were still dominant after the enzyme-triggered morphological transition as revealed from CD analyses (Figure 4d).

Figure 4. AFM height images of A9K2 self-assembled nanostructures a) before and b) after addition of 20 U/mL PAO. c) TEM image of A9K2 self-assembled nanostructures after addition of 20 U/mL PAO. d) CD spectra of the A9K2 solution of 8 mM before and after addition of 20 U/mL PAO. Before 21



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addition of PAO, the peptide solution was aged for 24 h at room temperature, followed by enzymatic incubation at 37 oC for 2 weeks.

The above results indicated that the enzymatically-formed long entangled nanofibers were microscopically responsible for the hydrogelation of A9K2. It is well known that LO and PAO can catalyze the oxidation of the primary amines of lysines to aldehydes, i.e. lysines to allysines.26, 33-35, 38 As shown in Figure 2, MS measurements indicated the formation of intra- and intermolecular lysinorleucines upon addition of PAO, which are the products of a series of sequential reactions between allysines and lysines, including condensation and reduction (Figure S2). Because the positively charged lysine residues are mostly located on the A9K2 self-assembled nanostructures’ surface, the formation of intermolecular lysinorleucines raises the possibility that the occurrence of long nanofibers might be a result of the enzymatic cross-linking of short nanorods via a head-to-tail mode. If this were true, we should also observe some thick bundles of nanorods/nanofibers upon addition of PAO, in which side-by-side cross-linking dictated. However, we only observed thin nanofibers, and furthermore, their heights showed little variation in comparison with those of nanorods (Figure 4 and Figure S3). These results rule out this possibility and the alignment of short nanorods into the long nanofibers is mostly likely to arise from other factors. According to RP-HPLC analysis (Figure S4), there was a reduction of approximately 33.3% in the A9K2 peak area after incubation with PAO for 2 weeks, indicating that about 1/3 of A9K2 molecules were transformed. A direct outcome of this enzymatic 22


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transformation of A9K2 molecules was that their lysine side chain charges were reduced, for example, a 50% reduction in the case of intra-molecular lysinorleucines. Therefore, the surface charge density of A9K2 self-assemblies (nanorods and micelles) should decrease upon addition of PAO or LO, thus causing the electrostatic repulsions between them to decrease. In order to trigger peptide self-assembly or hydrogelation, reducing the electrostatic repulsive forces of charged motifs either via adjusting solution pH or by increasing ionic concentration has been widely used, in which lowering the extent of side-chain ionization or electrostatic screening has been observed to induce the formation of nanofibers,39 to dramatically increase fiber length,40 and to transform spherical micelles into nanofibers.41 Therefore, we expect that the enzymatic attenuation of the electrostatic repulsions between A9K2 micelles and nanorods makes them align into nanofibers, eventually resulting in the hydrogelation of the A9K2 solution. To confirm this hypothesis, we increased the ionic strength of the peptide solution at pH 7.4 via the addition of NaCl. When the NaCl concentration was increased to 100 mM, hydrogelation occurred (Figure S5a), and AFM imaging indicated the formation of some nanofibers, which were less in amount and shorter in length, relative to the nanofibers produced by enzymatic means (Figure S5b). As a result, the gel formed in the presence of 100 mM NaCl displayed a storage modulus (Gʹ) of around 100 Pa (Figure S5c), significantly lower than that of the enzymatic gel (Figure 3). In spite of these minor differences, we reason that the enzyme-triggered gelation of the A9K2 solution arises from the enzymatic transformation of A9K2 molecules and the consequent formation of nanofibers. The 23



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enzymatic transformation reduces the electrostatic repulsions between A9K2 micelles and nanorods, thus causing their alignment into long nanofibers. Although A9K has a slightly higher antibacterial ability than A9K2,21,

22

the A9K

solution at 8 mM, and even at higher concentrations, was unable to form hydrogels upon addition of FBS or PAO and instead, it readily turned into opaque suspensions. This implies that the solubility of A9K and its assemblies has significantly decreased after the enzymatic oxidation of lysine to allysine. 3.2. Antibacterial Activity and Cytotoxicity of A9K2 Hydrogels We have shown that A9K2 could effectively inhibit bacterial growth at concentrations as low as 0.1 mg/mL whilst it displayed low toxicity against mammalian cells.22 In the present study, the enzymatic A9K2 hydrogel also showed effective antibacterial activity against both G- bacterial strains (E. coli and P. aeruginosa) and G+ strains (S. aureus and B. subtilis). We followed the viability of bacteria in the solution above the hydrogel surface and of those at the hydrogel/liquid interface after 24 h incubation. The bacteria were introduced at a concentration of 1×106 CFU/mL, which far exceeded the threshold inoculum for an infection associated with biomedical devices (< 100 CFUs).11 As shown in Figure 5a, there was negligible bacterial growth in the solution above the hydrogel from the four strains, in sharp contrast to the control in which bacterial growth in solution was substantial. Furthermore, Figure 5b shows that most of the bacteria in the solution above the hydrogel were stained red rather than green while their counterparts in the control mostly exhibited green fluorescence. 24


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Figure 5c indicates that under static culture conditions, there were rather higher levels of bacterial adhesion on the substrate irrespective of the presence of the peptide hydrogel. However, the bacteria on the hydrogel surface exhibited red rather than green fluorescence, whilst the ones on the control TCPS plate surface were well stained green. These results indicate that bacterial growth both in the solution above the A9K2 hydrogel and on the gel surface are strongly inhibited, in spite of a high inoculum, suggesting its high antibacterial activity. A9K2 has been demonstrated to kill bacteria via a mechanism of membrane permeabilization.22 PI only penetrates cells with damaged membranes and interacts with DNA and RNA,17, 28 so the observation that the bacteria in the presence of the A9K2 hydrogel were well stained by PI reveals that the peptide hydrogel induced bacterial death via a mechanism involving cell membrane destabilization. Furthermore, dead bacteria were prone to aggregation (Figure 5b), also suggesting a mechanism of membrane permeabilization.21 As indicated above, only part of A9K2 molecules was enzymatically transformed. During the 24 h incubation with bacteria, HPLC analysis indicated that 0.276 mg/mL of A9K2 diffused into the solution above the gel. It is thus possible that soluble A9K2 molecules which diffuse into the solution contribute to the observed antibacterial activity. In a study of the antibacterial activity of a β-hairpin peptide hydrogel, however, Salick et al suggested that the observed antibacterial activity was established by the gel surface.31 In this case, we observed that the majority of bacteria were concentrated on the hydrogel surface and killed. Thus, it is most likely that the combination of both diffused A9K2 25



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molecules and the A9K2 hydrogel surface is responsible for the observed antibacterial activity.

Figure 5. a) Bacterial growth in the solution above the A9K2 hydrogel surface after incubation for 24 h. b) Fluorescence microscopy images of the bacteria (B. subtilis 168) in the solution above the A9K2 hydrogel surface after incubation for 24 h. The bacteria were subject to a live/dead staining with SYTO 9 and PI prior to imaging. c) Confocal laser scanning microscopy images of the bacteria (B. subtilis 168) on the A9K2 hydrogel surface after incubation for 24 h and live/dead staining with SYTO 9 and PI, indicating most of the bacteria are deposited on the gel surface. In all these cases, the bacteria incubated in the TCPS plates in the absence of A9K2 gels acted as a control.

Considering A9K2 inhibited bacterial growth, possibly via a mechanism of membrane permeabilization, the cytotoxic effect of the peptide hydrogel was assessed by performing 26


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the LDH assay rather than the MTT assay. The latter measures the reduction of MTT into formazan by the redox activity of viable cells, and a decrease in MTT reduction can act as an index of cell death.22, 42, 43 Because the purple-colored MTT formazan is insoluble, dimethyl sulfoxide (DMSO) is usually introduced to dissolve the precipitated dye after removing the culture medium. In this case, some of the peptide gel was observed to exist in the resulting formazan-DMSO solution and significantly interfered with the subsequent absorbance measurement. In contrast, the LDH assay measures the intracellular enzyme LDH release into the culture medium upon damage of plasma membranes, and the activity of LDH released from dead cells can therefore act as an index of cellular death.42-45 In this case, because the LDH released was well dissolved in the aqueous medium, it could be well transferred to another culture plate and the method thus avoided possible interference from the peptide gel. When NIH 3T3 cells were cultured on the A9K2 hydrogel surface, there was little LDH release, with the LDH activity being even lower than the activity of the cells incubated on the control TCPS plate (Figure 6a). Simultaneously, the peptide also showed little LDH release when incubated with HEK293 cells for 72 h even at a much higher concentration of 16 mM (Figure S6). These results suggested an extremely low cytotoxicity of A9K2 and the peptide hydrogel towards mammalian cells, in spite of direct contact of the cells with the gel surface as well as some diffusion of A9K2 into the solution. LDH release showed an increasing trend with incubation time, a result of natural programmed cell death during this period.

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Soluble cytokines IL-8 and TNFα are major signaling molecules in the immune system, which are produced in response to infection.46 The two cytokines secreted by human lymphocytes were studied using the ELISA method.27,

32

As shown in Figure 6b, the

lymphocytes seeded on the A9K2 gel surface secreted low levels of TNFα and IL-8 after 48 h incubation, similar to those cultured in the control TCPS wells in the absence of the gel. However, the lymphocytes treated with 2 µg/mL LPS produced significant amounts of TNFα and IL-8, approximately 100 times higher than did the ones on the A9K2 hydrogel and the control plate. These results revealed that A9K2 hydrogel does not cause nonspecific immunogenic responses, being indicative of good biocompatibility.

Figure 6. a) LDH activity of NIH 3T3 cells plated on the A9K2 hydrogel and onto TCPS plates (control) as a function of incubation time. b) Concentrations of TNF-α and IL-8 secreted by lymphocytes plated on TCPS plates (control) and the A9K2 hydrogel after 48 h incubation, and by the LPS-treated lymphocytes.

Finally, co-culturing experiments were performed to further assess the selectivity of the A9K2 hydrogel. After the introduction of B. subtilis 168, NIH 3T3 cells in the control wells without the peptide hydrogel mostly became rounded and their nuclei stained bright 28


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red (PI), whilst the bacteria showed green fluorescence (SYTO 9), as shown in Figure 7a. These results indicated that the introduction of bacteria led to significant mammalian cell death in the absence of antibiotic within the culture medium. In contrast, a change in mammalian cell morphology was not observed when NIH 3T3 cells grown on the A9K2 hydrogel were exposed to B. subtilis 168. As shown in Figure 7b, the mammalian cells spread well on the gel surface and were well stained by calcein-AM (green), whilst the bacteria showed red fluorescence (PI), indicative of high cellular selectivity of the A9K2 hydrogel and avoidance of bacterial contamination.

Figure 7. Laser scanning confocal microscopy images of NIH 3T3 cells after the introduction of B. subtilis 168: a) in the TCPS plate without the A9K2 hydrogel and b) on the A9K2 hydrogel surface. Mammalian cells were stained with calcein-AM and PI, and bacterial cells were stained with SYTO 9 and PI. After the introduction of the bacteria, the co-culture system was incubated for 24 h. The scale bar is 10 µm.

Owing to the advantages of the A9K2 hydrogel outlined above, including its low cytotoxicity and good biocompatibility, high antibacterial activity and excellent cellular 29



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selectivity, it has great potential as a scaffold material for practical applications such as cell culture, regenerative medicine and cell/drug delivery. In order to transfer the material into clinical use, our future research will focus on its antibacterial behaviors under dynamic culture conditions, as well as the inhibition of bacterial adhesion and subsequent biofilm formation on the gel surface.

4. CONCLUSIONS The amphiphilic peptide A9K2 can self-assemble into micelles and short nanorods, and it demonstrates effective bactericidal properties. We demonstrated that the A9K2 solution underwent a sol-gel transition upon addition of FBS or PAO, accompanied by the formation of long entangled nanofibers. The transformation of A9K2 molecules catalyzed by LO in PBS or PAO and the consequent attenuation of the electrostatic repulsions between A9K2 micelles and nanorods are mechanistically responsible for the formation of nanofibers and the sol-gel transition of the A9K2 solution. Bacterial growth both in the solution above the A9K2 hydrogel and at the gel surface can be strongly inhibited via a mechanism involving membrane permeabilization. Furthermore, the enzymatic A9K2 hydrogel showed extremely low mammalian cell toxicity and did not induce nonspecific immunogenic responses in human lymphocytes. Under co-culturing conditions, the peptide hydrogel displayed excellent selectivity by favoring the adherence and spread of mammalian cells, whilst killing pathogenic bacteria, and is therefore capable of reducing bacterial contamination. These advantages make the enzymatic A9K2 hydrogel an ideal scaffold material for cell culture, regenerative medicine and drug delivery. 30


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ASSOCIATED CONTENT Supporting Information Photographs of the A9K2 aqueous solution after addition of denatured PAO and in the presence of inactivator of PAO; possible reaction pathways for the PAO-triggered formation of inter-molecular lysinorleucine and intra-molecular lysinorleucine from A9K2; AFM height section profiles of A9K2 self-assembled nanostructures before and after addition of PAO; RP-HPLC profiles of A9K2 before and after addition of PAO; photographs, AFM images and rheological properties of the A9K2 aqueous solution after addition of NaCl; LDH activity of HEK293 cells incubated with A9K2 for 72 h. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Authors E-mail: [email protected] (H.X); [email protected] (J.R.L.) Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS This work was funded by the National Natural Science Foundation of China under grant numbers 21373270 and 31271497, and the Fundamental Research Funds for the Central Universities (14CX02189A).

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REFERENCES (1) Lee, K. Y.; Mooney, D. J. Hydrogels for Tissue Engineering. Chem. Rev. 2001, 101, 1869-1879. (2) Cushing, M. C.; Anseth, K. S. Hydrogel Cell Cultures. Science 2007, 316, 1133-1134. (3) Slaughter, B. V.; Khurshid, S. S.; Fisher, O. Z.; Khademhosseini, A.; Peppas, N. A. Hydrogels in Regenerative Medicine. Adv. Mater. 2009, 21, 3307-3329. (4) Collier, J. H.; Rudra, J. S.; Gasiorowski, J. Z.; Jung, J. P. Multi-Component Extracellular Matrices Based on Pepide Self-Assembly. Chem. Soc. Rev. 2010, 39, 3413-3424. (5) Matson, J. B.; Stupp, S. I. Self-Assembling Peptide Scaffolds for Regenerative Medicine. Chem. Commun. 2012, 48, 26-33. (6) Wu, E. C.; Zhang, S.; Hauser, C. A. Self-Assembling Peptides as Cell-Interactive Scaffolds. Adv. Funct. Mater. 2012, 22, 456-468. (7) Jung, J. P.; Gasiorowski, J. Z.; Collier, J. H. Fibrillar Peptide Gels in Biotechnology and Biomedicine. Biopolymers 2010, 94, 49-59. (8) Mirjalili, A.; Parmoor, E.; Bidhendi, S. M.; Sarkari, B. Microbial Contamination of Cell Culture: A 2 Years Study. Biologicals 2005, 33, 81-85. (9) Merritt, K.; Hitchins, V. M.; Neale, A. R. Tissue Colonization form Implantable Biomaterials with Low Numbers of Bacteria. J. Biomed. Mater. Res. 1999, 44, 261-265. (10) Schierholz, J. M.; Beuth, J. Inplant Infections: A Haven for Opportunistic Bacteria. J. Hosp. Infect. 2001, 49, 87-93.

32


Page 32 of 38

Page 33 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(11) Edmiston, C. E.; Seabrook, G. R.; Goheen, M. P.; Krepel, C. J.; Johnson, C. P.; Lewis, B. D.; Brown, K. R.; Towne, J. B. Bacterial Adherence to Surgical Sutures: Can Antibacterial-Coated Sutures Reduce the Risk of Microbial Contamination? J. Am. Coll. Surg. 2006, 203, 481-489. (12) Edmiston, C. E.; Seabrook, G. R.; Cambria, R. A.; Brown, K. R.; Lewis, B. D.; Sommers, J. R.; Krepel, C. J.; Wilson, P. J.; Sinski, S.; Towne, J. B. Molecular Epidemiology of Microbial Contaminaiton in the Operating Room Environment: Is There a Risk for Infection? Surgery 2005, 138, 573-582. (13) Merritt, K.; Shafer, J. W.; Brown, S. A. Implant Site Infection Rates with Porous and Dense Materials. J. Biomed. Mater. Res. 1979, 13, 101-108. (14) Hancock, R. E. W.; Sahl, H.-G. Antimicrobial and Host-Defense Peptides as New Anti-Infective Therapeutic Strategies. Nat. Biotech. 2006, 24, 1551-1557. (15) Yeaman, M. R.; Yount, N. Y. Mechanisms of Antimicrobial Peptide Action and Resistance. Pharmacol. Rev. 2003, 55, 27-55. (16) Zasloff, M. Antimicrobial Peptides of Multicellular Oganisms. Nature 2002, 415, 389-395. (17) Chen, C.; Hu, J.; Zeng, P.; Chen, Y.; Xu, H.; Lu, J. High Cell Selectivity and Low-Level Antibacterial Resistance of Designed Amphiphilic Peptide G(IIKK)3I-NH2. ACS Appl. Mater. Interfaces 2014, 6, 16529-16536. (18) von Maltzahn, G.; Vauthey, S.; Santoso, S.; Zhang, S. Positively Charged Surfactant-Like Peptides Self-Assemble into Nanostructures. Langmuir 2003, 19, 4332-4337.

33



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(19) Xu, H.; Wang, J.; Han, S.; Wang, J.; Yu, D.; Zhang, H.; Xia, D.; Zhao, X.; Waigh, T. A.; Lu, J. R. Hydrophobic-Region-Induced Transitions in Self-Assembled Peptide Nanostructures. Langmuir 2009, 25, 4115-4123. (20) Makovitzki, A.; Baram, J.; Shai, Y. Antimicrobial Lipopolypeptides Composed of Palmitoyl Diand Tricationic Peptides: In vitro and in vivo Activities, Self-Assembly to Nanostructures, and a Plausible Mode of Action. Biochemistry 2008, 47, 10630-10636. (21) Chen, C.; Pan, F.; Zhang, S.; Hu, J.; Cao, M.; Wang, J.; Xu, H.; Zhao, X.; Lu, J. Antibacterial Activities of Short Designer Peptides: A Link Between Propensity for Nanostructuring and Capacity for Membrane Destabilization. Biomacromolecules 2010, 11, 402-411. (22) Chen, C.; Hu, J.; Zhang, S.; Zhou, P.; Zhao, X.; Xu, H.; Zhao, X.; Yaseen, M.; Lu, J. Molecular Mechanisms of Antibacterial and Antitumor Actions of Designed Surfactant-Like Peptides. Biomaterials 2012, 33, 592-603. (23) Yang, Z.; Liang, G.; Xu, B. Enzymatic Hydrogelation of Small Molecules. Acc. Chem. Res. 2008, 41, 315-326. (24) Zelzer, M.; Todd, S. J.; Hirst, A. R.; McDonald, T. O.; Ulijn, R. V. Enzyme Responsive Materials: Design Strategies and Future Developments. Biomater. Sci. 2013, 1, 11-39. (25) Gao, Y.; Yang, Z.; Kuang, Y.; Ma, M.-L.; Li, J.; Zhao, F.; Xu B. Enzyme-Instructed Self-Assembly of Peptide Derivatives to Form Nanofibers and Hydrogels. Biopolymers 2010, 94, 19-31. (26) Kagan, H. M.; Li, W. Lysyl Oxidase: Properties, Specificity, and Biological Roles Inside and Outside of the Cell. J Cell. Biochem. 2003, 88, 660-672. 34


Page 34 of 38

Page 35 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(27) Chen, C.; Gu, Y.; Deng, L.; Han, S.; Sun, X.; Chen, Y.; Lu, J. R.; Xu, H. Tuning Gelation Kinetics and Mechanical Rigidity of β-Hairpin Peptide Hydrogels via Hydrophobic Amino Acid Substitutions. ACS Appl. Mater. Interfaces 2014, 6, 14360-14369. (28) Boulos, L.; Prévost, M.; Barbeau, B.; Coallier, J.; Desjardins, R. LIVE/DEAD®BacLightTM: Application of a New Rapid Staining Method for Direct Enumeration of Viable and Total Bacteria in Drinking Water. J. Microbiol. Methods 1999, 37, 77-86. (29) Biggerstaff, J. P.; Puil, M. L.; Weidow, B. L.; Prater, J.; Glass, K.; Radosevich, M.; White, D. New Methodology for Viability Testing in Environmental Samples. Mol. Cell. Probes 2006, 20, 141-146. (30) Alonso, J. L.; Mascellaro, S.; Moreno, Y.; Ferrús, M. A.; Hernández, J. Double-Staining Method for Differentiation of Morphological Changes and Membrane Integrity of Campylobacter coli Cells. Appl. Environ. Microbiol. 2002, 68, 5151-5154. (31) Salick, D. A.; Kretsinger, J. K.; Pochan, J. D.; Schneider, J. P. Inherent Antibacterial Activity of a Peptide-Based β-Hairpin Hydrogel. J. Am. Chem. Soc. 2007, 129, 14793-14799. (32) Chen, C.; Hu, J.; Zeng, P.; Pan, F.; Yaseen, M.; Xu, H.; Lu, J. Molecular Mechanisms of Anticancer Action and Cell Selectivity of Short α-Helical Peptides. Biomaterials 2014, 35, 1552-1561. (33) Bakota, E. L.; Aulisa, L.; Galler, K. M.; Hartgerink, J. D. Enzymatic Cross-Linking of a Nanofibrous Hydrogel. Biomacromolecules 2011, 12, 82-87. (34) Suva, R. H.; Abeles, R. H. Studies of the Mechanism of Action of Plasma Amine Oxidase. Biochemistry 1978, 17, 3538-3545.

35



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 36 of 38

(35) Shah, M. A.; Scaman, C. H.; Palcic, M. M.; Kagan, H. M. Kinetics and Stereospecificity of the Lysyl Oxidase Reaction. J. Biol. Chem. 1993, 268, 11573-11579. (36) Hevey, R. C.; Babson, J.; Maycock, A. L.; Abeles, R. H. Highly Specific Enzyme Inhibitors. Inhibition of Plasma Amine Oxidase. J. Am. Chem. Soc. 1973, 95, 6125-6127. (37) Rucker, R. B.; Kosonen, T.; Clegg, M. S.; Mitchell, A. E.; Rucker, B. R.; Uriu-Hare, J. Y.; Keen, C. L. Copper, Lysyl Oxidase, and Extracellular Matrix Protein Cross-Linking. Am. J. Clin. Nutr. 1998, 67, 996s-1002s. (38) Teixeira, L. S. M.; Feijen, J.; van Blitterswijk, C. A.; Dijkstra, P. J.; Karperien, M. Enzyme-Catalyzed Crosslinkable Hydrogels: Emerging Strategies for Tissue Engineering. Biomaterials 2012, 33, 1281-1290. (39) Hartgerink, J. D.; Benisash, E.; Stupp, S. I. Peptide-Amphiphile Nanofibers: A Versatile Scaffold for the Preparation of Self-Assembling Materials. Proc. Natl. Acad. Sci. U. S. A. 2002, 99, 5133-5138. (40) Dong, H.; Paramonov, S. E.; Aulisa, L.; Bakota, E. L.; Hartgerink, J. D. Self-Assembly of Multidomain Peptides: Balancing Molecular Frustration Controls Conformation and Nanostructture. J. Am. Chem. Soc. 2007, 129, 12468-12472. (41) Ghosh, A.; Haverick, M.; Stump, K.; Yang, X.; Tweedle, M. F.; Goldberger, J. E. Fine-Tuning the pH Trigger of Self-Assembly. J. Am. Chem. Soc. 2012, 134, 3647-3650. (42)

Abe,

K.;

Matsuki,

N.

Measurement

of

Cellular

3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) Reduction Activity and Lactate Dehydrogenase Release Using MTT. Neurosci. Res. 2000, 38, 325-329.

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(43) Izak-Nau, E.; Kenesei, K.; Murali, K.; Voetz, M.; Eiden, S.; Puntes, V. F.; Duschl, A.; Madarász, E. Interaction of Differently Functionalized Fluorescent Silica Nanoparticles with Neural Stem- and Tissue-Type Cells. Nanotoxicology 2014, 8, 138-148 (44) Sepp, A.; Binns, R. M.; Lechler, R. L. Improved Protocol for Colorimetric Detection of Complement-Mediated Cytotoxicity Based on the Measurement of Cytoplasmic

Lactate

Dehydrogenase Activity. J. Immunol. Methods 1996, 196, 175-180. (45) Choksakulnimitr, S.; Masuda, S.; Tokuda, H.; Takakura, Y.; Hashida, M. In vitro Cytotoxicity of Macromolecules in Different Cell Culture Systems. J. Controlled Release 1995, 34, 233-241. (46) De, G. D.; Zangerle, P. F.; Gevaert, Y.; Fassotte, M. F.; Beguin, Y.; Noizat-Pirenne, F.; Pirenne, J.; Gathy, R.; Lopez, M.; Dehart, I. Direct Stimulation of Cytokines (IL-1β, TNF-α, IL-6, IL-2, IFN-γ and GM-CSF) in Whole Blood. I. Comparison with Isolated PBMC Stimulation. Cytokine 1992, 4, 239-248.

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Enzymatic Regulation of Self-Assembling Peptide A9K2

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