Insights into the Antimicrobial Activity and Cytotoxicity of Engineered Î±

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Insights into the Antimicrobial Activity and Cytotoxicity of Engineered #-Helical Peptide Amphiphiles Zhi Ma, Jing Yang, Jinzhi Han, Ling Gao, Hongxia Liu, Zhaoxin Lu, Haizhen Zhao, and Xiaomei Bie J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00922 • Publication Date (Web): 28 Nov 2016 Downloaded from http://pubs.acs.org on November 28, 2016

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Journal of Medicinal Chemistry

Insights into the Antimicrobial Activity and Cytotoxicity of Engineered α-Helical Peptide Amphiphiles Zhi Ma, Jing Yang, Jinzhi Han, Ling Gao, Hongxia Liu, Zhaoxin Lu, Haizhen Zhao, and Xiaomei Bie* Key Laboratory of Food Processing and Quality Control, Ministry of Agriculture of China, College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, People’s Republic of China

*Correspondence: College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, China. E-mail: [email protected] (X. Bie)

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ABSTRACT: Antimicrobial peptides (AMPs) have gained increasing attention, as they can overcome recurring microbial invasions. However, their poor antimicrobial activity and potential cytotoxicity remain impediments to their clinical applications as novel therapeutic agents. To enhance the antimicrobial activity and cell selectivity of AMPs, a series of amphiphilic peptides based on Leucocin A were designed by substituting non-charged hydrophilic residues with arginine and leucine. Of the engineered peptides, peptide 7 (WRL3) (WLRAFRRLVRRLARGLRR-NH2) exhibited the highest cell selectivity towards bacterial cells over erythrocytes and macrophages. Fluorescent measurements and microscopic observations demonstrated that 7 increased cell membrane permeability and disrupted membrane envelope integrity, and eventually led to whole cell lysis. Additionally, flow cytometry analysis and sub-cellular localization studies revealed that 7 showed potent cytotoxicity against human hepatoma cells HepG2. In summary, the data indicate that these engineered peptides, in particular 7, have enormous promise for antibacterial and/or antitumor therapeutics. KEYWORDS: Antimicrobial peptide; a-Helix; Amphipathicity; Antibacterial activity; Cytotoxicity.


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■ INTRODUCTION Antibiotic resistance has been a global health concern due to the indiscriminate use of antibiotics and the subsequent creation of bacteria that can survive traditional treatment.1 Therefore, identification of novel antibiotic therapies is needed to better treat current resistant infections and to decrease the likelihood of future resistance to susceptible bacterial species.2 Antimicrobial peptides (AMPs) are endogenous components of the innate immune response that exist in various organisms and have been shown to be clinical candidates for new drugs3, 4 Different from conventional antibiotics that target certain biosynthetic pathways crucial to cell wall or protein synthesis, the majority of AMPs kill bacteria by physically penetrating membranes and initiating lysis, enabling the leakage of nutrients and essential ions and thereby reducing the possibility of developing bacterial resistance.5 By exploiting this mechanism, peptidebased antimicrobials could be useful for diverse biomedical applications, such as nanomaterials, cosmetics, anticancer agents and biomedical coatings.6 Presently, more than 2,000 AMPs have been isolated and identified from various organisms (e.g., bacteria, plants, animals) and have been documented to possess antibacterial, antifungal, anticancer and immuno-modulatory activities.7, 8 Several AMPs are in different clinical trial phases, but many technological hurdles remain, such as antimicrobial activity optimization, cell selectivity enhancement and reduced hemolytic activity.4 Unfortunately, the rational design of peptide structures has some limitations and these contribute to differences in efficacy among the designed drugs. Positive charge, hydrophobicity and amphipathicity are considered essential for AMP functions, even though the mode of action is elusive. Cationic charges are helpful for the peptides to bind the negatively charged bacterial membranes and hydrophobic moieties of peptides possibly provide lipophilic anchors that eventually cause membrane disruption. However, these variables have no stringent relation to antibacterial activity. Even so, a balanced amphipathicity is widely accepted to be critical for maximizing antibacterial activity while minimizing the cytotoxicity of AMPs.9 Leucocin A, produced by Leuconostoc gelidum UAL 187, exhibits high cell specificity and no hemolytic activity, but possesses a narrow antimicrobial spectra against only Gram-positive bacteria and low antimicrobial potency.10 Here, we describe the systematic design of a series of amphiphilic peptides, in which uncharged hydrophilic residues were substituted with arginine and leucine along the C-terminal α-



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helical region of Leucocin A (18-35). The incorporation of Arg residues into the engineered peptides is aimed at exploiting Arg’s high charge density and improving antimicrobial activities.11 A net positive charge exists in the side chain of Arg, which is pivotal for adhering to the negatively-charged outer layer of bacterial membranes. Furthermore, molecular dynamics simulations have verified that Arg-rich AMPs are more easily inserted into the bacterial membrane when helped by membrane thinning, and the formation of Arg-water hydrogen and Arg-phosphate hydrogen bonds.12 As previously reported, Leu residues have a strong tendency to adopt an amphipathic helix. Therefore, introduction of Leu can both increase hydrophobicity and contribute to maintaining the helical structure of the designed peptides.13 We speculate that these modifications would make the engineered peptides have a high cell selectivity and low hemolysis while increasing antimicrobial potency and cytotoxic effects against tumor cells. The secondary structures of these engineered peptides were identified using circular dichroism (CD) in membrane-like milieu, including trifluoroethyl alcohol (TFE) and sodium dodecyl sulfate (SDS). The antibacterial activity of the peptides was then evaluated with or without the addition of physiological salts via minimum inhibitory concentration (MIC) assays against various infectious microorganisms, including Staphylococcus aureus (Gram-positive), Escherichia coli (Gram-negative) and Candida albicans (yeast). The underlying cytotoxicity of these engineered peptides was investigated with erythrocyte hemolysis, with the bactericidal efficiency evaluated with a time-kill kinetic assay. The potential mechanisms of membrane destruction were studied by measuring membrane potential changes and outer/inner membrane permeabilization, along with scanning electron microscopy (SEM), transmission electron microscopy (TEM) and flow cytometry. Moreover, to investigate whether the synthetic peptides possessed lipopolysaccharide (LPS)-neutralizing activities, BODIPY-TR-cadaverine (BC) substitution and LPS-neutralizing assays were also conducted. Whether the molecular mechanism(s) by which some AMPs target and kill tumor cells are similar to their antimicrobial properties is unclear, but the electrostatic attractions between AMPs and target membranes are considered to be a critical step for both activities.14 As tumor cell has a negatively charged lipid membrane,15 we assessed AMP cytotoxicity against human hepatoma HepG2 cells using flow cytometry, cell morphology and sub-cellular localization assays to investigate the potential for developing these peptides as novel antimicrobial and/or antitumor agents.


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■ RESULTS Peptide Design and Characterization. Matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy (MALDI-TOF MS) was used to confirm peptide molecular weights, with the theoretically calculated and measured molecular weights overviewed in Table 1. The measured molecular weight of each peptide concurred with the theoretical values. Moreover, the data of 1H nuclear magnetic resonance (NMR) spectra were consistent with the structure of these peptides (Supporting Information Figure S2). These results indicated we obtained the desired compounds. Dissimilarities in the peptide hydrophobicity in water-based mixture corresponded to the various HPLC retention times. The hydrophobic characteristics for peptides and their retention times were summarized in Table 1. The wheel-diagram and the 3D structure projection results demonstrated that the positively-charged residues of the seven engineered peptides were found on one face, while the hydrophobic residues were found on the opposite face, representing a highly amphipathic structure (Figure 1 and Figure 2). Relative hydrophobic moments of the designed peptides were consecutively enhanced from 0.451 to 0.869 (Table 1), showing an improved equilibrium among the hydrophobicity and hydrophilicity compared the parental peptide 1 (WG18) (0.431). Secondary Structure of Peptides. CD spectroscopy was used to investigate the secondary structures of the engineered peptides (Supporting Information Figure S3), with the percentages of α-helix, β-sheet, turn and arbitrary coil computed in the different solutions (Table 2). CD spectra demonstrated that the engineered peptides in the deionized water showed the disordered conformations with a negative peak near 198 nm. By comparison, in SDS/TFE, two negative dichroic bands were observed in the CD spectra at about 208 and 222 nm, indicating the α-helical characteristic for the engineered peptides. In addition, the propensity for an α-helical conformation in 1, 6 (WRL2), 7 and 8 (WRL4) in the membrane-like milieu seemed to be enhanced in contrast to that of 2 (WR1), 3 (WR3), 4 (WR5) and 5 (WR7). This was confirmed by their α-helical contents in SDS and TFE solutions (Table 2). Hemolytic Activity and Cytotoxicity. Peptide hemolytic activities were summarized in Table 3 and Supporting Information Figure S4. Even at the highest peptide concentration of 64 µM, all tested peptides exhibited minor or no erythrocyte hemolysis, with the exception of melittin, which caused the



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maximal hemolysis of 63.73%. Next, the peptide cytotoxicity against RAW264.7 cells was determined through dose-response studies (Figure 3). The cell viabilities induced by 1, 2, 3, 4, 5, 6, 7 and 8 at 64 µΜ were 73.62%, 77.58%, 87.22%, 85.48%, 74.80%, 64.79%, 40.73% and 48.25%, respectively, while melittin eliminated the majority of the living cells, with the lowest viability measured at 1.85%. Antimicrobial and Selective Activities. Peptide antimicrobial activities were evaluated against various clinical pathogens, such as E. coli, S. aureus and C. albicans. As revealed in Table 3, the engineered peptides showed wide antibacterial spectra against a series of microbes, with geometric mean (GM) MICs ranging from 0.46 to 142.3 µM. Overall, peptide 7 displayed the greatest antibacterial activity, with GM MICs of 0.46~1.32 µM, comparable to melittin (GM = 0.8 µM). Compared with polymyxin B, peptide 7 showed broader antibacterial spectra against Gram-positive and Gram-negative bacteria, although polymyxin B had higher antibacterial activity against Gram-negative bacteria. The therapeutic index (TI), which is often utilized to assess the cell selectivity of novel compounds, was calculated as the ratio of minimal hemolysis concentration (MHC) to GM of MIC values. As shown in Table 3, 7 displayed the greatest TI (20.53~59.15), which was an improvement of > 20-fold compared to the parent peptide 1 (TI=1). 5, 6 and 8 had lower TIs of 7.51~8.0, 10.29~11.76, and 4.83~5.88, respectively. The data demonstrated that 7 was greater selective towards microorganisms over erythrocytes, suggesting a larger treatment window. Resistance to antibiotics and AMPs has been confirmed to be an essential component of pathogenesis in some resistant bacteria and has been constructed in laboratory evolutionary experiments.16 In this study, all E. coli variant selection lines displayed significant increases in MIC values compared with the control selection lines (Supporting Information Table S1). The E. coli variants evolved higher tolerance to gentamicin than to peptide 7 during continued selection (Supporting Information Table S1). Moreover, the E. coli variants induced by gentamicin had higher MIC50 (the concentration of a drug that reduced bacterial growth by 50% or more) values after selection than those induced by 7 (Supporting Information Figure S5). Overall, the use of peptide 7 resulted in reduced occurrence of bacterial resistance in comparison with gentamicin. The hemolytic and antibacterial activities indicate that these engineered peptides, in particular 7, can be used as potential lead compounds in the development of new antimicrobial agents. Therefore, the ACS Paragon Plus Environment

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kinetics of the peptides’ interaction with the bacteria was further investigated (Supporting Information Figure S6). All engineered peptides induced time-dependent growth inhibition against both bacterial species. Peptide 7 eliminated almost all E. coli cells (Supporting Information Figure S6A) and S. aureus cells (Supporting Information Figure S6B) within 5 min at 1 × MIC, indicating a substantial and rapid killing efficacy. Although similar trends were observed for the other peptides, their killing efficiencies were markedly decreased. Salt Sensitivity. To study the effects of cations on antibacterial activity of the designed peptides, the MIC values of the engineered peptides against S. aureus and E. coli cells were measured with the addition of several salts. As shown in Table 4, the addition of monovalent (Na+, NH4+ and K+) and divalent cations (Mg2+ and Ca2+) had little effect or even promoted bacteriostatic activities of the peptides, with the exceptions of 3 and 8 which lost most antibacterial activity against E. coli in the presence of Na+, Ca2+ and Mg2+. The most significant loss in antibacterial activity was observed for all peptides in the existence of trivalent Fe3+. Of the designed peptides, 7 was the most tolerant to the presence of physiological salts. Outer and Inner Membrane Permeabilization. The peptides’ potential to permeabilize bacterial outer membranes was studied with an N-phenyl-1-naphthylamine (NPN) uptake assay. When the outer membrane is disrupted and functionally impaired, NPN is dissociated from the disturbed outer membrane and shows improved fluorescence. As shown in Supporting Information Figure S7A, all the engineered peptides improved the permeability of outer membrane dose-dependently, with the exception of 1, which did not induce an increase in NPN fluorescent intensity even at the highest concentration of 7.5 µM. In contrast, peptide 7 demonstrated the highest potency in the permeability of the outer membrane. Next, inner membrane permeability was assessed by an o-nitrophenol-β-D-galactoside (ONPG) assay. When the inner membrane of E. coli is damaged, ONPG may infiltrate into the cytoplasm, in which it can be cleaved to ONP by the intracellular β-galactosidase, and the ONP is then free from E. coli into the medium. Thus, the concentration of ONP in the culture medium is an indicator of the inner membrane permeabilization.17 As shown in Supporting Information Figure S7B, the potency of all peptides to permeabilize E. coli inner membrane was weak, except for peptide 7, which induced a remarkable increase in inner membrane permeability, similar to that induced by melittin.



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Cytoplasmic

Membrane

Electrical

Potential.

The

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bacterial cytoplasmic membrane

depolarization caused by the peptides was determined with 3, 3’-dipropylthiadicarbocyanine iodide (diSC35), which was a membrane potential-dependent fluorescent dye. Once the bacterial cytoplasmic membrane was disturbed and damaged by the peptides, the cytoplasmic membrane electrical potential would be dissipated and diSC3-5 would be released into the medium, resulting in a subsequent enhancement in fluorescence intensity. Peptide 7 and melittin rapidly increased fluorescence, while the other peptides caused only a slight membrane depolarization (Supporting Information Figure S8). Overall, the increased fluorescence after treatment with 7 at 2 × MICs was more significant than that observed after treatment with 1 × MICs and 0.5 × MICs. Flow Cytometry. Considering the antibacterial activities and the membrane permeabilization capacities, peptides 1 and 7 were chosen for additional evaluations for the antimicrobial mechanisms by determining the integrality of bacterial membrane after treatment with peptides (Figure 4). Propidium iodide (PI) marks fluorescently the DNA of cells after bacterial membrane destruction. With no peptide treatment, 94.3% of the bacterial cells demonstrated no PI fluorescence, suggesting intact bacterial cytoplasmic membranes (Figure 4A). However, after 1 × MIC peptide 7 treatment for 30 min, more than 90% of cells were PI-stained. The same experiment with peptide 1 in place of 7 resulted in the staining of only 22.9% of cells. In general, both of the peptides 1 and 7 dose-dependently destroyed the bacterial membranes, but 7 induced a higher increase in the PI fluorescence than 1 did. Microscopic Observations. In order to further elucidate antimicrobial mechanisms of the engineered peptides against S. aureus 29213 and E. coli O157:H7 cells, SEM was implemented to study morphological alterations. With no peptide treatment, the bacterial cells showed brilliant and smooth membrane surfaces (Figure 5). However, after treatment with peptides at 1 × MIC, the membrane stunting, creping and destruction were found in the S. aureus and E. coli cells. Compared to peptide 1-treated bacteria, the bacterial membranes after treatment with peptide 7 showed more roughening with additional blebbing. TEM manifested the morphologic and intracellular changes in E. coli and S. aureus cells after treatment with peptides 1 and 7. In the absence of peptide, the bacterial cells retained smooth surfaces and


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dense internal structures (Figure 6). After 60 min of treatment, peptide 1 induced minor alterations in bacterial membrane morphology and intracellular changes. In comparison, peptide 7 induced substantial damage of bacterial membranes and outflow of the cytoplasm. Confocal laser-scanning microscope (CLSM) was used to further investigate the site targeted by fluorescein isothiocyanate (FITC)-labelled 1 and 7 in E. coli O157:H7. As shown in Figure 7, compared with peptide 1, bacterial cells treated with peptide 7 presented green rectangular rings with fluorescent signal spread across the bacterial membrane surfaces, indicating that 7 targeted the bacterial cell membrane. LPS-Neutralizing Activities. To determine the binding affinity of the synthetic peptides to LPS, the BC replacement assay was conducted (Figure 8). Overall, compared with the parental peptide 1, the engineered peptides caused stronger dose-dependent increases in fluorescent intensity. 7 demonstrated the greatest effect, similar to that of polymyxin B, which is a positive control for binding LPS. Thus, peptide 7 effectively binds to LPS on the E. coli outer membrane. The endotoxin-neutralizing abilities of the designed peptides were determined by provoking RAW264.7 cells with 500 ng/mL LPS in the existence of various peptides. The amounts of two typical inflammatory mediators, tumor necrosis factor (TNF)-α and nitric oxide (NO), were measured in cell culture supernatants. As shown in Figure 9A, the engineered peptides suppressed NO production in a concentration-dependent manner. Compared with 1, peptide 7 displayed the highest LPS-neutralizing activity, with a peptide concentration of 32 µM reducing the nitrite concentration to the control level (no LPS treatment). A similar trend for the inhibition of TNF-α production was observed with the engineered peptides (Figure 9B). Interestingly, peptide 7 appeared to potentiate the neutralization of anionic LPS when suppressing TNF-α generation, with noticeably reduced TNF-α levels at a lower concentration of 4 µM. Cytotoxicity against HepG2 Cells. Peptide cytotoxicity against HepG2 cells was measured using the 3-(4, 5-dimethylthiozol-2-yl)-2, 5- diphenyltetrazolium bromide (MTT) assay. The survival rates of HepG2 cells over a peptide concentration range of 0.25-64 µM were measured, with the engineered peptides showing cytotoxicity that was dose-dependent (Figure 10). The IC50 (the concentration inducing tumour cell death by 50%) of peptide 7 against HepG2 cells was ~32 µM, displaying the greatest growthinhibitory effect (Supporting Information Table S2). Overall, the peptides reduced the viability of tumor cells similar to the prior bactericidal activity (Supporting Information Figure S6). ACS Paragon Plus Environment


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Morphological Characterization. Inverted microscopy and SEM were used to assess morphologic alterations in the treated cells. After 32 µM peptide 7 treatment for 24 h, the amount of HepG2 cells persisting as an adhesive monolayer was remarkably decreased in contrast to the control cells (Figure 11A and E). SEM imaging revealed that untreated HepG2 cells showed a normal smooth surface and spread morphology (Figure 11B, C and D), whereas 7-treated cells displayed roughened or inhomogeneous cell membranes (Figure 11F, G and H). As shown in Figure 11F, 7-treated HepG2 cells had crimped boundaries and a rounded appearance. In Figure 11G and H, 7-treated HepG2 cells were heavily disrupted and shriveled. Additionally, a pore (indicated by the arrow) was clearly visible on the membrane surface, indicating potential cytoskeleton damage. Thus, peptide 7 appears to kill HepG2 cells via membrane disruption and subsequent leakage of cytoplasmic contents. Cell Cycle and Apoptosis. The influence of peptides on cell cycle and apoptosis progression in HepG2 was investigated by PI staining and flow cytometry. According to previous studies,18 DNA content is a marker of cellular maturity in the cell cycle: DNA content of cells in the G0/1 phase is set to 1 unit of DNA; cells reproduce DNA in the S phase, improving their content proportionate to progress through S; and upon starting G2 and later M phases, cells have double G0/1 phase DNA content (2 units). During apoptosis, a portion of nuclear DNA may be lost from the cell in shedding apoptotic bodies and some fragmented DNA may be extracted during fixation and staining. Consequently, apoptotic cells are often distinguished by their fractional DNA content, namely “sub-G1” cells. In many experiments, cells are immobilized with the alcohol and then stained with a nucleic acid-specific fluorescent dye, PI. The PI radiates red fluorescence at 488 nm (excitation) when bound to DNA, the differences in fluorescence intensity reflect the proportions of cells in the respective phase of cell cycle. As shown in Table 5 and Figure 12, peptide treatments resulted in a great aggregation of the cells in the sub-G1 phase, together with a substantial lowering in the G1 phase in the HepG2 cells, compared with control cells. Noticeably, peptide 7 (64 µM) significantly increased the percentage of sub-G1 cells (77.6%), suggesting that peptide 7 induced prodigious apoptosis, especially at a higher concentration (Table 5 and Figure 12E). Nuclear Morphological Change and Peptide Location. To directly observe the changes in the nuclei of apoptotic cells induced by the engineered peptides, 4’, 6-diamidino-2-phenylindole (DAPI)


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assay was performed. As shown in Figure 13, in the absence of peptide, the dim fluorescence spread on the surface of cells indicated that most DAPI did not penetrate into the viable cells and bind to the nucleus. However, following treatment with 32 µΜ peptide 7 for 2 and 4 h, nuclei (blue) could be visualized by DAPI staining. The nuclei were notably condensed, and small parts of the nucleus bud off could be observed. After 8 h of peptide treatment, a large number of nuclear fragments were scattered throughout the cytoplasm (Figure 13). To precisely locate the engineered peptides during incubation with HepG2 cells, FITC-7 was used at a concentration of 32 µM and its fluorescence distribution was visualized using CLSM at different points during the incubation period. As shown in Supporting Information Figure S9, after 1 h of incubation, the majority of FITC-derived signals (green) were located on the cell membranes (indicated by the arrows) and relatively weak fluorescent signals were observed within the cytoplasm, indicating that the membranes surrounding the cells were the primary targets during this period. After 4 h of incubation, fluorescent signals clustered in the nucleus, suggesting the translocation of the peptide across the outer lipid bilayers and subsequent interaction with the negatively charged DNA molecules. After 8 h of treatment, the green fluorescence was distributed throughout the cell. Furthermore, the cell boundary became blurry presumably due to the gradual structural deterioration of cell membranes during this process. Measurement of Reactive Oxygenic Species (ROS) Production. As shown in Figure 14, peptide 7 induced dose-dependent and time-dependent increases in the 2', 7'-dichlorofluorescin (DCFH) fluorescence intensity in HepG2 cells (Figure 14 A and B). The increase in the fluorescence was visual at 2 h (Figure 14B). However, there was no change in the fluorescence intensity induced by peptide 1 (Figure 14A). Overall, an increase in ROS production could be induced by peptide 7 in HepG2 cells. ■ DISCUSSION AND CONCLUSION AMPs have received widespread attention due to their broad antimicrobial spectra against Gram-negative and positive bacteria, viruses, fungi, multiple drug-resistant bacteria, parasites, and even tumor cells. Most AMPs eradicate the microorganisms by irreversibly destroying cell membranes, and this distinctive mechanism substantially lowers the incidence of pathogens. Synthetic biomaterials, however, are often poorly biocompatible, inducing ectopic cytotoxicity, significant immunogenicity and reduced antibacterial activity. In order to avoid these intrinsic restrictions, a number of optimized designs, including ACS Paragon Plus Environment


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glycosylation,19 fluorination,20 cyclization,21 hybridization,22 lipidation,23 phosphorylation24 and the insertion of D-amino acids

25

have been performed, whereas these models are both complex and expensive. A

simple scientific rationale has yet to be developed for designing optimized AMPs. Amphipathicity is considered the most important structural parameter for the activity of AMPs. In the present study, we designed and synthesized a series of highly amphiphilic peptides with Arg and Leu substitutions, as shown in Figure 1 and Figure 2. The antibacterial assay confirmed that the designed peptides were more efficient, compared with the parental peptide 1 (Table 3), probably because of the increases in net charge and amphiphilicity (Table 1). As previously reported,26 an increase in the positive charge favored electrostatic interaction of AMPs to the negatively-charged components of bacterial membranes, thus enhancing antimicrobial activity and killing efficacy (Table 3 and Supporting Information Figure S6). However, peptide 5 did not exhibit greater antimicrobial activity than peptide 4, although it has more net charges (+9) compared to 4 (+7). Therefore, the charge is not positively correlated with the antibacterial activity of the peptides and it seems to exist a threshold. For short peptides (10-20 residues), the enhancement of cationic charges over a threshold value (usually +6) would not improve antibacterial activity.11 In this study, the number of net charges of 4, 5, 6, 7 and 8 was all beyond +6, indicating adequate electrostatic attraction of these engineered peptides to the negatively-charged membranes and a weaker charge-activity connection. To a certain extent, the hydrophobicity promoted the antimicrobial potential of AMPs,27 which may explain why 6, 7 and 8 have both greater hydrophobic values and stronger antimicrobial activities than 5. Whereas hydrophobicity has no simply linear relation to the antibacterial activity. The retention time (RT) typically reflects the hydrophobicity of the engineered peptide. In this study, even though peptide 8 displayed the largest hydrophobicity (RT=16.98 min), its antimicrobial activity was relatively lower compared to that of peptide 7 (11.62). Perhaps a hydrophobic threshold value pertinent to membrane damage potential of the peptide may elucidate this phenomenon, such that a further increase in hydrophobicity beyond the threshold value, as found in peptide 8, deteriorates the antimicrobial activity due to the steric hindrance effect among the hydrophobic residues.25 Based on these results, an appropriate amphipathy may play the most critical role in the antimicrobial activity of these engineered peptides. Given the wheel-diagram and 3D structure projection results (Figure 1 and Figure 2), Arg and Leu substitutions significantly increased the propensity for an amphiphilic helical formation, which correlated well with


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inhibition of bacterial growth (Table 3). Many researches have shown that the tendency to fold an amphiphilic α-helical structure in membrane-mimetic surroundings is crucial to increasing peptide membrane-destructive activity.28 The toxicity of the synthetic peptides against the host cells was frequently believed to be the chief limitations in using AMPs as novel therapeutics. The engineered peptides displayed minimal or no hemolytic activity against erythrocytes at various MIC values (Supporting Information Figure S4), suggesting a great cell selectivity toward the negative bacterial membranes than zwitterionic mammalian cell membranes, likely due to the rationally-designed amphipathicity of the peptides. Notably, the toxicity of AMPs was frequently associated with the increased antibacterial activity attributed to homogenous determinant factors, including peptide hydrophobicity and helicity.29 Therefore, it is not difficult to infer that the engineered peptides, especially 7, had relatively greater hemolysis and cytotoxicity presumably attributed to the increase of hydrophobicity. Thus, an optimum hydrophobicity-amphiphilicity equilibrium may be a primary focus in the AMP designs. In the present study, some cations such as Na+, Ca2+ and Fe3+ compromised peptide antibacterial activities against Gram-positive and Gram-negative bacteria. Previous studies have shown that the presence of salts will weaken electrostatic effects among positively-charged peptides and negativelycharged membranes to block the cationic peptides attaching to the bacteria, leading to the reduced antibacterial activity.22 Cationic AMPs frequently displayed lowered activity in the presence of cations, but this was not always the case. The antibacterial activity of our engineered peptides was increased with the addition of certain salts (KCl, NH4Cl and MgCl2) (Table 4). Zhu et al. have demonstrated that the antimicrobial activity of RAW4, an amphipathic peptide derived from the cathelicidin PMAP-36, was increased by the addition of K+.30 Dong et al. have proposed that a relatively small number of divalent cations added will be helpful to the binding of AMPs to the bacterial membranes.11 Thus, the effects of cations depend on the peptides and their concentrations, a phenomenon that may explain why salt ions increased peptide activity in this study. Moreover, Fe3+ more significantly suppressed antibacterial activities of the peptides compared with monovalent and divalent cations. In previous studies, multivalent cations affected peptide antibacterial activities by impeding upon electrostatic interactions and triggering membrane-binding competing effects of cations and peptides.31



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Generally, the initial step of bacterial killing is the selective binding of cationic peptides to the negatively-charged components, such as hydroxylated phospholipids and LPS, of the bacterial membrane, followed by disruption of the organisms by perturbing membrane balance and forming a transmembrane pore, or penetrating into the cell, thus damaging the cellular machineries.4, 11 This could provide a rationale for the relatively minor alteration of the MIC values required for the engineered peptides in this study against MRSA and E. coli mutants (Table 3 and Supporting Information Table S1). To verify this hypothesis, the synthetic peptides, in particular 7, were demonstrated to cause permeabilization of the inner/outer membrane to the extent that small molecules such as fluorochrome NPN and ONPG are able to cross these barriers, suggesting a potent membrane-permeabilizing capacity. It has a large membrane potential (-130 to -150 mV) amid the intact bacterial membrane.30 The peptides will induce dissipation of this potential barrier by perturbing the bacterial membrane. 7 effectively depolarized the cytoplasmic membrane, indicating that potential dissipation may be correlated with the formation of ionic channels or transmembrane pores, thereby resulting in leakage of cytoplasmic contents. Flow cytometry indicated that 7 could potently damage the bacterial cell membrane, thereby allowing PI to bind nucleic acids. The fluorescence distribution shown by CLSM proved that peptide 7 targeted the bacterial membranes. SEM and TEM imaging additionally demonstrated that 7 caused significant membrane disruption, with the appearance of membrane disruption and cytosolic leakage. Another undesirable property of designed antimicrobials is related to the immunogenicity caused by the interaction of the synthetic peptides and the host cells via the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway,32, 33 which happens in hypercytokinemia or cytokine storm that induces serious inflammation. LPS, also known as endotoxins, are found in the outer membrane of Gram-negative bacteria and have potent immunostimulating properties, which activates Tolllike Receptor-4 (TLR4), followed by the nuclear factor kappa B (NF-КB) transcription factor in the monocytes, thereby leading to downstream release of inflammatory modulators like TNF-α and NO. Previous reports have demonstrated that certain cationic peptides, such as indolicidin34 and HBD-2,35 can decrease endotoxin-induced production of inflammatory mediators by inhibiting the interaction of LPS with LPS binding protein (LBP) or by directly binding to LPS. These features enable AMPs to be desirable therapeutics for Gram-negative caused endotoxin stress and septicemia therapies. In this study, 7 showed


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stronger LPS-neutralizing ability at concentrations ≥ 8 µM relative to parental peptide 1, which was corresponding to the results obtained from the BC replacement test. As reported previously,36 the positively-charged amphipathic AMPs can neutralize the negatively-charged amphipathic lipid A domains, which are widely considered as the valid moieties of endotoxins, by electrostatic interaction, further causing LPS polymers to dissociate due to hydrophobic interactions between the nonpolar side chains of AMPs and the alkyl chains of LPS.37 As the engineered peptides possessed a relatively higher hydrophobicity and net charge, it is not difficult to infer that the engineered peptides, especially peptide 7 (net charge of +9), showed greater anti-LPS activity. However, at the MIC or even 5-fold higher (usually used in animal infection models), peptide 7 did not show significant LPS neutralization. This case was consistent with the results reported by Ong et al.27 One theory that may explain this phenomenon was the existence of inorganic salts and matrix metalloproteinases (MMPs) secreted by the macrophages in the cell culture media, which would decrease peptide activities. DMEM medium used for cell culture contained a variety of salt ions, such as NaCl (6400 mg/L), KCl (400 mg/L), MgSO4 (97.67 mg/L) and CaCl2 (200 mg/L), these monovalent and bivalent cations would affect the LPS-neutralizing activity of the peptides by initiating LPSbinding competing effects of cations and peptides38 and interfering with electrostatic attractions between the cationic portion of the peptide and the anionic head group of LPS.11,

39

Moreover, the engineered

peptides would be partially hydrolyzed by MMPs, such as MMP-3 and MMP-9, which could degrade a number of proteins and peptides that constituted the extracellular matrix.40, 41 Hence, it is not difficult to understand why peptide 7 need a relatively higher concentration than MIC when efficiently neutralizing LPS. Moreover, cell viability exceeded 80% at 16, 64, 64 and 64 µM for 7, 3, 4 and 5, respectively (Figure 3). Therefore, peptide anti-inflammatory properties were independent of their effects on the cell viability and these engineered peptides were nontoxic, demonstrating their compatibility as novel therapeutic agents. Although different antimicrobial peptides exhibited various bactericidal effects or anti-inflammatory activities, only a select few were effective in killing tumor cells. The engineered peptide 7 concentrationdependently suppressed HepG2 cell proliferation and viability (Figure 10). SEM studies suggested that the primary mechanism of action for the killing of cancer cells by peptide 7 was through membrane disruption, and subsequent cell apoptosis. In fact, hypodiploid DNA content peak and ROS generation in 7-treated HepG2 cells, a hallmark of cell apoptosis,42 were markedly detected using flow cytometry and confocal



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microscopy. Moreover, sub cellular localization studies revealed that 7 could traverse cancer cell membranes after disrupting them and then interact with the intracellular targets, such as nuclei. These results indicate that 7 has a strong potency against tumor cells. As previously indicated, natural AMP, such as melittin, also displays antimicrobial and anticancer activities.43 However, it suffers from a number of drawbacks. In spite of having high antimicrobial and anticancer activities, melittin and its biomimetic analogs caused heavy hemolysis (Supporting Information Figure S4), suggesting low cell selectivity. peptide 7 was more selectively killing bacterial and tumor cells with less toxicity against erythrocytes and macrophages (Table 3, Figure 3 and Figure 10), together with its potent anti-inflammatory activity (Figure 9) as well as high physiological salt resistance (Table 4), potentially making it a promising therapeutic agents for future clinical applications. In conclusion, a series of amphiphilic α-helical peptides were designed based on Leucocin A using arginine and leucine substitutions. These amphiphilic peptides showed an disordered structure in the deionized water, but was coiled into an α-helical formation following exposure to the membrane-like surroundings. Through a series of in vitro molecular and cellular studies, these peptides, in particular 7, exhibited potent antibacterial activity and cytotoxicity against hepatoma cells while remaining low toxicity against host cells. These beneficial characters were mostly attributed to an appropriate equilibrium between cationicity and hydrophobicity and a well-engineered facially amphipathic configuration. Microscopic observations and fluorescent assays revealed that the engineered peptides killed microorganisms and tumor cells by destroying cell membranes, resulting in outflow of the cytoplasm and ultimately lysing the whole cell. Additionally, 7 exhibited significant endotoxin-binding and neutralizing capabilities. Thus, these synthetic peptides, in particular 7, have potential as novel antimicrobial/antitumor agents. While the data stated in this study show only in vitro effects and are still at an early stage of development, the rational design and optimizing of AMPs are helpful in additional evaluations of in vivo assays, with the final objective of utilizing the antimicrobial and antitumor potency in the biotechnological and clinical applications, including bio-nanotechnology, surfactant development, biomedicine and implantable devices. ■ EXPERIMENTAL SECTION


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Materials. Mueller-Hinton broth (MHB) powder, Mueller-Hinton Agar (MHA) and Potato Dextrose Agar (PDA) obtained from AoBoX (China) were used to incubate microbes according to the manufacturer’s instructions. Bovine serum albumin (BSA) and SDS were obtained from Sigma-Aldrich (USA), and TFE was purchased from Amresco (USA). BSA, SDS and TFE were used as dilution agents. Phosphatebuffered saline (PBS) solution obtained from Solarbio (China) was used after dilution to achieve the desired concentrations. Sodium chloride, potassium chloride, ammonium chloride, magnesium chloride, calcium chloride and ferric chloride, which were purchased from Kermel (China), were of analytical grade. Triton X100, NPN, diSC3-5, LPS from E. coli 0111:B4, HEPES, ethanol (analytical grade, >99%), acetone (analytical grade, >99%), tertiary butanol (analytical grade, >99%), and glutaraldehyde (synthetic grade, 50% in H2O) were all obtained from Sigma-Aldrich (China). Peptide Design and Sequence Analysis. The amphiphilic peptides were constructed by increasing the positive charges on the polar face and the hydrophobicity on the nonpolar face of the αhelical peptide 1, truncated from Leucocin A, with arginine (R) and leucine (L) substitutions, respectively. Firstly, 1, an 18-residue α-helical peptide, a truncated portion of the C-terminal region of Leucocin A (18-35), was obtained. This variable C-terminal helix was hypothesized to govern the specificity and spectrum of activity of the peptide.10 The wheel-diagram demonstrated that 1 possessed less net positive charges and an imperfectly amphiphilic structure, with a negatively-charged residue Glu (E) and several uncharged hydrophilic residues such as Asn (N), His (H), Gly (G) and Ser (S) on one face and a few hydrophobic residues Val (V), Phe (F), Leu (L) and Trp (W) on the opposite face (Figure 1). However, previous studies have shown that cationicity and amphipathicity can, to a large extent, affect the bactericidal activity of AMPs.27 Secondly, the amphipathic AMPs were designed based on the α-helical protein framework and folding principles. In order to enhance the net positive charges of 1, four peptides 2, 3, 4 and 5 were designed and synthesized with R substituted for E3, or E3, N14 and N17, or E3, A7, H10, N14 and N17, or E3, S6, A7, H10, N14, N17 and G18, respectively. These 1 derivatives were predicted to adopt an α-helical conformation without segregation of the nonpolar and polar side chains. To acquire a highly amphipathic structure, the hydrophobicity of 5 was increased. Three analogues 6, 7 and 8 of peptide 5 were designed and synthesized with L substituted for G8 and G16, or G2, G8 and G16, or G2, G8, G15 and G16. 6, 7 and 8 displayed extremely amphipathic characteristic, with positively-charged hydrophilic ACS Paragon Plus Environment


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residues Arg on the polar side chain and hydrophobic residues Val (V), Phe (F), Leu (L) and Trp (W) on the nonpolar side chain (Figure 1). The primary physicochemical parameters and sequences of the engineered peptides were calculated by

bioinformatics

programs,

including

http://www.expasy.org/tools/protparam.html)

and

ProtParam the

(ExPASy antimicrobial

Proteomics peptide

Server: database

(http://aps.unmc.edu/AP/main.php). The secondary structure content of the peptides was calculated online using the site: http://perry.freeshell.org/raussens.html. The three-dimensional structure projections were predicted online with I-TASSER (http://zhanglab.ccmb.med.umich.edu/I-TASSER/). The helical wheel projection was performed online using the Helical Wheel Projections: (http://heliquest.ipmc.cnrs.fr/). Synthesis and Characterization of Peptides. In this study, the engineered peptides were synthesized and purified by GL Biochem Corporation (Shanghai, China) through solid-phase methods using N-9-fluorenylmethyloxycarbonyl (Fmoc) chemistry. The true molecular weights were confirmed through MALDI-TOF MS (Bruker Daltonics Inc., USA) using α-cyano-4-hydroxycinnamic acid as the matrix. 1

H NMR spectra of the peptides were obtained on a Bruker Avance 500 MHz spectrometer at room

temperature (300 K). The chemical shifts were expressed in ppm with 3-(trimethylsilyl) propionic-2, 2, 3, 3d4 acid (TSP) as internal standards, and coupling constants (J) were reported in hertz (Hz). D2O was used as solvent for peptides 1-7, the solvent residual signal was found at a chemical shift of 4.68 ppm. For peptide 8, a 1:3 (v/v) CD3CN:D2O solution was used as the solvent, and the solvent residual peaks were found at 2.01 ppm and 4.37 ppm, respectively. Peptide purity was more than 95% according to analytical reverse-phase high-performance liquid chromatography (RP-HPLC) (Supporting Information Figure S1). Peptides were dissolved in deionized water and stored at -20 °C before the following structural and antibacterial assessments. CD Spectra of the Peptides. CD spectra of peptides were obtained at 25 °C with a J-820 spectropolarimeter (Jasco, Tokyo, Japan) equipped with a quartz cell with a path length of 0.1 cm. Spectra were recorded (λ190-250

nm)

at a peptide concentration of 100 µg/ml

in deionized water, 30 mM SDS

(environment comparable to a negatively-charged prokaryotic membrane), and 50% (v/v) TFE (mimicking the hydrophobic environment of the microbial membrane). An average of three scans was recorded for


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each peptide. The acquired CD signal spectra were then converted to the mean residue ellipticity using the following equation: θM = (θobs • 1000)/(c • l • n) Where θM is the mean residue ellipticity [deg.cm2.dmol-1], θobs is the observed ellipticity corrected for the buffer at a given wavelength [mdeg], c is the peptide concentration [mM], l is the path length [mm], and n is the number of amino acids. Antimicrobial Assays. E. coli ATCC 25922, E. coli ATCC 35150, Salmonella choleraesuis ATCC 13312, methicillin-resistant S. aureus (MRSA) ATCC 43300, S. aureus ATCC 29213, and yeast C. albicans ATCC 10231 were acquired from the American Type Culture Collection (ATCC). E. coli O157:H7 CICC 21530, Salmonella paratyphi-A CICC 20501, Salmonella paratyphi-B CICC 21495, Salmonella paratyphi-C CICC 21512, Salmonella enteritidis CICC 21527, Vibrio parahaemolyticus CICC 21617, Listeria monocytogenes CICC 21662, Listeria monocytogenes CICC 21633, Pseudomonas fluorescens CICC 21620 were obtained from the China Center of Industrial Culture Collection (CICC); Bacillus cereus CMCC 63301, Micrococcus luteus CMCC 28001, Bucillus Pumilus CMCC 63202, Shigella flexneri CMCC 51571, Salmonella typhimurium CMCC 51005 were purchased from the Chinese Medical Culture Collection (CMCC); S. aureus GIM 1.160 was from Guangdong Microbiology Culture Center (GIM). The MICs of the peptides were determined using a modified standard microtiter dilution method as previously described.44 The bacteria were incubated overnight at 37 °C and transferred to new nutrient broth (MHB) until the logarithmic phase of growth. The bacteria were then diluted to 105 colony forming units (CFU)/mL in MHB. The peptides were dissolved and diluted in 0.01% acetic acid and 0.2% BSA. A 50-µL volume of bacteria in MHB was mixed with 50-µL of serially diluted aliquots of the peptides in sterile 96-well plates. The mixtures were incubated at 37 °C for 18-24 h in an incubator. The MIC was determined by optical density (OD) measurements at a wavelength of 492 nm as the lowest concentration of the peptide that resulted in no bacterial growth. The results were expressed as an average of the MICs obtained from three to five independent experiments. To investigate whether resistance to our engineered peptides would evolve under continued selection, AMP and antibiotic-resistant variants of E. coli O157:H7 were developed by step-wise culture at 37 °C in MHB with selected peptide 7 and gentamicin, hereafter referred to as E. coli variant selection lines.



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Moreover, we also established E. coli O157:H7 lineages growing in an environment devoid of 7 and gentamicin for the entire duration of the experiment, here referred to as E. coli control selection lines. All lines were initially grown in a pristine environment free of 7 and gentamicin. At transfer 1, we supplemented the media of the variant selection lines with a non-inhibitory concentration of 7 (1 µM) and gentamicin (0.06 µM), as shown in Supplemental Table S1. Afterwards, we doubled the concentration every 2 transfers until transfer 10. After that, we doubled the concentration every 10 transfers until transfer 100 (about 600–700 bacterial generations). Every time we increased the concentration of antimicrobials, a sample of each line was inoculated to 25% (v/v) glycerol and stored at -80 °C. We assayed the resistance evolved by measuring MICs of 7 and gentamicin for each selection line, using bacterial samples of transfer 10, transfer 50 and transfer 100. The growth of these cultures was then measured in a serial dilution of 7 and gentamicin concentrations using a microplate reader at 492 nm after incubation for 18 h. The MIC50 of each culture was estimated as the concentration of 7 or gentamicin that reduced bacterial growth by 50% or more.45 The kinetics of bacterial killing was assessed using E. coli O157:H7 and S. aureus 29213. Bacteria grown to the logarithmic phase were separately exposed to each peptide at 1 × MIC at 37 °C in 10 mM PBS (pH 7.4) for 0-90 min. Aliquots were collected at fixed intervals, diluted and plated on nutrient agar plates. The number of colonies was counted after incubation at 37 °C for 18 h. The results were expressed as an average of the data from three independent assays. Measurement of Hemolytic Activity. Peptide hemolytic activity was measured according to the published methods.27 Fresh human red blood cells (hRBCs) were obtained from healthy donors and collected in a sterile heparinized borosilicate tube, and then centrifuged immediately at 1,000 × g and 4 °C for 5 min. The obtained erythrocytes were washed three times with 10 mM PBS (pH 7.4), and then resuspended in PBS to attain a dilution of approximately 1% (v/v) erythrocytes (5.4 x 108 cells/ml). Briefly, a 50-µL of the hRBC solution was incubated with 50-µL of different peptide concentrations dissolved in PBS for 1 h at 37 °C. Mixtures were centrifuged at 2,000 × g and 4 °C for 5 min. 60-µL of each supernatant was then transferred to another new 96-well microtiter plate. The release of hemoglobin was monitored by measuring the absorbance at 540 nm using a multi-mode microplate reader (SpectraMax M5, Molecular Devices, USA). The negative control (0% hemolysis) consisted of hRBCs in PBS only, and the positive


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control (100% hemolysis) consisted of hRBCs in 0.1% Triton X-100 only. Peptide concentration that caused 10% hemolysis was considered to be the minimal hemolysis concentration (MHC). Percent hemolysis was calculated using the following formula: Percent hemolysis = [(A − A0) / (At − A0)] × 100 Where A represents the absorbance of the peptide sample at 540 nm, A0 and At represent 0% and 100% hemolysis determined in 10 mM PBS and 0.1% Triton X-100, respectively. Salt Sensitivity Assays. To determine whether the antimicrobial activity was affected by the presence of salts,46 we determined the MICs in MHB supplemented with different salts at their physiological concentrations (150 mM NaCl, 4.5 mM KCl, 6 mM NH4Cl, 1 mM MgCl2, 2 mM CaCl2 and 4 mM FeCl3), as described above. The results shown were from three independent assays. Outer and Inner Membrane Permeability Assay. The outer membrane permeability activity against bacteria was determined by the uptake of NPN, a ﬂuorescent dye that is sensitive to the outer membrane, as described previously.47 Briefly, E. coli O157:H7 grown to the logarithmic phase (OD600 = 0.4) at 37 °C were harvested by centrifugation at 2,000 × g and 4 °C for 10 min. The cells were washed twice with washing buffer (5 mM HEPES and 5 mM glucose, pH 7.4) and re-suspended to OD600 = 0.2. NPN was added to 2 ml of the bacterial suspension in a quartz cuvette to a final concentration of 10 µM. Aliquots of the peptides were then added to the bacterial suspension. The background fluorescence was recorded with an LS-50B fluorescence spectrophotometer (Perkin Elmer, USA). The excitation and emission wavelengths were set to 350 nm and 420 nm, respectively, and the fluorescence was recorded as a function of time until no further increase in the fluorescence was observed. Polymyxin B was used as a positive control. The inner membrane permeabilization of E. coli O157:H7 in the presence of peptides at their 1 × MICs was assessed through the measurement of β-galactosidase activity using ONPG, a substrate for cytoplasmic β-galactosidase.17 In brief, mid-log phase E. coli cells were diluted to an OD600 of 0.05 with 10 mM PBS (pH 7.4, containing 1.5 mM ONPG). The hydrolysis of ONPG to o-nitrophenol (ONP) was used to indicate the permeability state of the inner membrane by monitoring the absorbance at 420 nm over time, following the addition of peptides.



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Cytoplasmic Membrane Depolarization Assay. The membrane depolarization activities of the peptides were determined using E. coli O157:H7 and a membrane potential-sensitive ﬂuorescent dye, diSC3-5, as previously described.48 Briefly, E. coli cells were grown to log phase, harvested by centrifugation at 2,000 × g and 4 °C for 10 min, washed twice with washing buffer (5 mM HEPES and 20 mM glucose, pH 7.4) and re-suspended to an OD600 of 0.05 in the same buffer. The cell suspension was incubated with 0.4 µM diSC3-5 for 90 min such that most of the dye molecules gathered at the ＋

cytoplasmic membrane. To equilibrate the cytoplasmic and external K concentrations, KCl was added to the cell suspension containing diSC3-5 to obtain a final concentration of 100 mM, followed by incubating the cells at room temperature for 15-30 min, with peptide aliquots added to 2 ml of suspension. The fluorescence was recorded using a model LS-50B fluorescence spectrophotometer at an excitation wavelength of 622 nm and an emission wavelength of 670 nm. Flow Cytometry. Membrane damage was determined by flow cytometry.49 Briefly, E. coli O157:H7 cells were grown to mid log phase in MHB, washed three times with PBS and diluted to 105 CFU/mL in the same buffer. The peptides 1 and 7 at their 0.5 × MICs, 1 × MICs and 2 × MICs were incubated with the bacterial suspension at a fixed PI concentration of 10 µg/mL for 30 min at 4 °C, followed by the removal of unbound dye by washing with an excess of PBS. Readings were obtained with a FACS flow cytometer (Becton-Dickinson, USA) at a laser excitation wavelength of 488 nm. SEM and TEM Characterization. E. coli O157:H7 and S. aureus 29213 cells were grown to the exponential phase in MHB at 37 °C under constant shaking at 220 rpm. After centrifugation at 2000 × g for 10 min, the cell pellets were harvested, washed twice with PBS, and re-suspended to an OD600 of 0.2. The cells were incubated at 37 °C for 60 min with different peptides at their 1 × MICs, with no peptide added to the control. After incubation, the cells were harvested via centrifugation at 5000 × g for 5 min, washed three times with PBS, fixed overnight with 2.5% glutaraldehyde at 4 °C, and washed twice with PBS. The cells were dehydrated for 10 min in a graded ethanol series (50, 70, 90, and 100%), followed by 15 min in 100% ethanol, a mixture (1:1) of 100% ethanol and tertiary butanol, and absolute tertiary butanol. The specimens were then dehydrated, dried, coated with gold, and examined using a HITACHI S-4800 SEM.


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Bacterial sample preparation for TEM was conducted using the same protocol as that for SEM. Bacterial cells were pre-fixed with 2.5% glutaraldehyde at 4 °C overnight, washed twice with PBS, and post-fixed with 2% osmium tetroxide for 70 min. After washing twice with PBS, the bacterial samples were dehydrated for 8 min in a graded ethanol series (50, 70, 90, and 100%), followed by 10 min in 100% ethanol, a mixture (1:1) of 100% ethanol and acetone, and absolute acetone. The specimens were then transferred to 1:1 mixtures of absolute acetone and epoxy resin for 30 min and then to pure epoxy resin and incubated overnight at a constant temperature. Specimens were then sectioned using an ultramicrotome, stained with uranyl acetate and lead citrate, and examined using a HITACHI H-7650 TEM.50 CLSM. E. coli O157:H7 cells (107 CFU/ml) were incubated with FITC-labelled peptides 1 and 7 at 1 × MIC for 30 min, and the bacterial cells were washed three times with PBS with centrifugation at 1,000 × g for 10 min. A smear was made, and the images were captured using a Leica TCS SP2 confocal laserscanning microscope with a 488-nm band-pass filter for the FITC excitation.51 Cytotoxicity Assay. Cell Culture: The murine macrophage cell line RAW264.7 was purchased from the Institute of Biochemistry and Cell Biology, SIBS, CAS (Shanghai, China) and maintained in DMEM medium (Gibco) supplemented with 100 U/mL penicillin and 100 mg/mL streptomycin (1% P/S, Invitrogen), 2 mM L-glutamine, and 10% fetal bovine serum (FBS, Gibco). Cells were cultured at 37 ºC in a humidified atmosphere of 5% CO2 and 95% air and passaged every 2-3 days. RAW264.7 cytotoxicity was determined using the colorimetric MTT dye reduction assay. Briefly, cells were seeded at a density of 1.0-2.0 × 104 cells/well in a 96-well plate and then treated with the peptides at final concentrations of 0.25-64 µM. After incubation for 24 h at 37 °C, cell cultures were further incubated with MTT (50 µl, 0.5 mg/mL) for 4 h at 37°C, centrifuged at 1,000 × g for 5 min and the supernatants discarded. Subsequently, 150 µl of dimethyl sulfoxide was added to dissolve formazan crystals, and the OD values were measured using a microplate reader (TECAN, Switzerland) at 570 nm.46 LPS Binding Assay. The peptide binding affinities to LPS were examined using a fluorescent dye BC (Sigma, USA) displacement assay, where BC fluorescence was quenched by LPS binding and alleviated by LPS/peptide binding. In a quartz cuvette, an equal volume of peptide solutions was added to a



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Page 24 of 52

mixture (2 ml) of LPS (25 µg/mL) and BC (2.5 µg/mL) in tris buffer (pH 7.4), and the background fluorescence was recorded (excitation wavelength 580 nm, emission wavelength 620 nm). The changes in fluorescence were recorded with a LS-50B fluorescence spectrophotometer, with each test performed independently in triplicate. Melittin was used as a positive control.21 Endotoxin Neutralization Assay. RAW264.7 cells were plated at a density of 1-2 × 105 cell/mL and stimulated with LPS (500 ng/mL) in the presence (0.5-64 µM) or absence of peptides in each well of a 96-well plate. Untreated cells and LPS-only treated cells served as negative and positive controls, respectively. Following incubation at 37 °C for 24 h, the supernatants were removed for analysis of TNF-α concentrations by ELISA (Sigma-Aldrich, USA) and NO levels using Griess reagent (1% sulfanilamide, 0.1% N-1-napthylethylenediamine dihydrochloride, 5% phosphoric acid) according to the manufacturer’s protocols.52 Cytotoxicity of the Peptides against Hepatoma Cells. Cell Culture: Human hepatoma cell line HepG2 was obtained from the SIBS (Shanghai, China). The cells were cultured in high-glucose DMEM (Gibco), 10% FBS and 1% P/S at 37 °C in a humidified atmosphere containing 5% CO2 passaged every 1-2 days. The cytotoxicity of the peptides against HepG2 was assessed using the MTT assay following the above method. The cells were incubated for 24 h with the peptides (0.25-64 µM) and for 4 h with MTT (50 µl, 0.5 mg/mL). The medium from each well was discarded and the resulting formazon crystals were solubilized by adding 150 µl of dimethyl sulfoxide. The OD was measured using a microplate reader at 570 nm. The MTT assay allowed the determination of IC50 from the dose-response curves using multiple dose of each peptide and Doxorubicin. IC50 was defined as the concentration of drug needed to inhibit cell growth by 50% relative to the untreated cells.53 Cell Morphology Analysis. In order to investigate the morphology changes of peptide treated tumor cells, HepG2 cells were pre-seeded on a coverslip at a density of 2 × 105 cells/mL for 24 h at 37 °C in a 5% CO2 atmosphere. The peptide solution was then added to a final concentration of 32 µM, and


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incubated for a further 24 h. The coverslips containing cells without peptides served as the control. The cells were observed by using an inverted microscope and SEM. Cell Cycle and Apoptosis Analysis. The cell cycle and apoptosis analysis was performed as described previously.18 A density of 2 × 105 cells/well was seeded into 6-well plates and left to attach overnight. The cells were incubated with 0, 32, 64 µM of the peptides for 24 h. After incubation, both floating and attached cells were harvested and washed with 10 mM PBS, re-suspended and fixed in 70% ice-cold ethanol overnight at 4 °C. Then cells were then treated with the cell cycle and apoptosis analysis kit (Beyotime, China) and the fluorescence intensity was measured using a FACS flow cytometer (BectonDickinson, USA). The cell cycle distribution and apoptosis were analyzed with FlowJo software. DAPI Staining and Sub-cellular Localization. To examine the changes in the nuclei of HepG2 cells undergoing apoptosis, DAPI staining was performed as reported previously.54 Briefly, HepG2 cells (1×105) were cultured with 32 µΜ peptide 7 for 0, 2, 4 and 8 h, and then incubated with 100 ng/ml DAPI for 15 min at 37 °C and washed with PBS (pH 7.4) six times. The fluorescence imaging was performed using a Leica TCS SP2 CLSM. HepG2 cells were pre-seeded in a 6-well plate overnight and then treated with FITC-7 at a final concentration of 32 µΜ for 0, 1, 4 and 8 h. After that, the cells were gently washed with PBS thrice and observed by confocal microscopy. Measurement of ROS Production. The intracellular generation of ROS was measured by 2', 7'dichlorofluorescin diacetate (DCFH-DA). The non-fluorescent ester penetrated into the cells and was hydrolyzed to DCFH by cellular esterases. The probe was rapidly oxidized to the highly fluorescent compound DCF in the presence of cellular peroxidase and ROS such as hydrogen peroxide or fatty acid peroxides. Briefly, HepG2 cells (1×105) were treated with peptides 1 and 7 at 0, 2, 4, 8, 16 and 32 µΜ for 8 hours, and loaded with 10 µΜ DCFH-DA for 30 min at 37 °C in the dark, then washed with PBS (pH 7.4) five times. The fluorescence was measured using an LS-50B fluorescence spectrophotometer and CLSM at an excitation of 485 nm and an emission of 525 nm. Statistical Analysis. All experiments were carried out on three independent occasions unless otherwise stated. Data are expressed as the mean ± standard deviations (SD). Statistical comparisons



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were performed using the ANOVA (t-test) with SAS 9.2 software. Differences with a value of P < 0.05 were considered statistically significantly. ■ASSOCIATED CONTENT Supporting Information Table S1: The evolved resistance of E. coli O157:H7 to the peptide and antibiotic; Table S2: The cytotoxicity of the designed peptides and Doxorubicin against HepG2 cells; Figure S1: HPLC spectrum of the synthetic peptides; Figure S2: 1H NMR spectra of the peptides; Figure S3: The CD spectra of the peptides; Figure S4: Hemolytic activity curves of the peptides; Figure S5: Growth of E. coli O157:H7 in relation to concentrations of 7 and gentamicin; Figure S6: Time-kill kinetic curves of the peptides; Figure S7: The outer and inner membrane permeability of the peptides; Figure S8: The cytoplasmic membrane potential variation of peptide-treated E. coli O157:H7; Figure S9: Sub-locations of FITC-7 in HepG2 cells using confocal microscopy. ■ AUTHOR INFORMATION *

Corresponding Author

For Xiaomei Bie, Telephone, +86 25 84396570; E-mail, [email protected]; Postal address: 1 Weigang, Xuanwu District, Nanjing, China. Author Contributions Z. Ma and X.M. Bie conceived and designed this research, J. Yang, J.Z. Han, L. Gao and H.X. Liu performed the experiments, Z.X. Lu and H.Z. Zhao reviewed and edited the manuscript. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest. ■ ACKNOWLEDGMENTS This study was supported by the National Science and Technology Support Program (Grant No. 2012BAK08807), the Social Development Program of Jiangsu Province (Grant No. BE2012746) and the Independent Innovation Program of Jiangsu Province (Grant No. CX (12)3087). ■ ABBREVIATIONS USED


Page 27 of 52

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AMPs, antimicrobial peptides; hRBCs, human red blood cells; LPS, lipopolysaccharides; MIC, minimum inhibitory concentration; BC, BODIPY-TR-cadaverine; SDS, sodium dodecyl sulfate; TFE, trifluoroethyl alcohol; GM, geometric mean; TI, therapeutic index; TLR4, Toll-like Receptor-4; LBP, LPS-binding protein; RP-HPLC, reverse-phase high-performance liquid chromatography; ONPG, o-nitrophenol-β-D-galactoside; MTT, 3-(4, 5-dimethylthiozol-2-yl)-2, 5- diphenyltetrazolium bromide.



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

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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


Table 1. Peptide designs and their structural information. a

b

Peptides

CN

Sequence

AA

NC

WG18

1

WGEAFSAGVHRLANGGNG-NH2

18

1

1798.91

WR1

2

WGRAFSAGVHRLANGGNG-NH2

18

3

WR3

3

WGRAFSAGVHRLARGGRG-NH2

18

WR5

4

WGRAFSRGVRRLARGGRG-NH2

WR7

5

WRL2

c

d

e

RT (min)

H

µHrel

1798.95

16.19

0.29

0.43

1825.90

1826.03

12.41

0.26

0.45

5

1910.13

1910.19

9.71

0.22

0.49

18

7

2014.32

2014.35

9.58

0.08

0.61

WGRAFRRGVRRLARGGRR-NH2

18

9

2182.52

2182.60

9.53

-0.03

0.66

6

WGRAFRRLVRRLARGLRR-NH2

18

9

2294.75

2294.81

11.52

0.16

0.80

WRL3

7

WLRAFRRLVRRLARGLRR-NH2

18

9

2350.81

2350.92

11.62

0.26

0.84

WRL4

8

WLRAFRRLVRRLARLLRR-NH2

18

9

2406.96

2407.03

16.98

0.35

0.87

a

b

c

Theoretical MW Measured MW

d

Compound number. Net charge. Molecular weight (MW). Retention time (RT) (min). HPLC retention

time was determined using a 4.6 × 250 mm Venusil MP C18-5 column (Waters, Milford, MA, USA) at a wavelength of 220 nm with a linear acetonitrile gradient from 15% to 45% in 30 min with buffer A (0.1% trifluoroacetic acid in acetonitrile) and buffer B (0.1% trifluoroacetic acid in water) at a flow rate of 1 mL/min. e

Hydrophobicity (H) and fhydrophobic moment (µHrel) of the peptides were calculated by HeliQuest.


f


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 34 of 52

Table 2. Quantification estimation of different secondary structures of the designed peptides. Peptides

Water

SDS

TFE

Helix

Beta

Turn

Random

Helix

Beta

Turn

Random

Helix

Beta

Turn

Random

1

0

12.9

26.4

60.7

60.7

17.4

0

21.9

63.1

0

0

36.9

2

0

14.7

25.3

60

44.7

27.8

0

27.5

26.5

32.7

0

40.8

3

0

17.7

25.7

56.6

37.5

34.4

0

28.1

50.4

6.2

0

43.4

4

5.9

47.5

12

34.6

17.1

42.5

0

40.4

38.1

15.1

0

46.9

5

0

0

31.1

68.9

13.2

50.7

0

36.2

39.1

21.5

0

39.4

6

7

25.2

21.4

46.4

42.2

25.6

0

32.2

54.9

6.4

0

38.7

7

0

27.5

17.4

55.1

48.3

22

0

29.7

49.6

12.7

0

37.7

8

0

17.3

22.6

60.1

62.1

14.8

0

23.1

55.2

5.8

0

39


Page 35 of 52


1 2 Table 3. Antibacterial and hemolytic activities of the peptides and antibiotics. 3 a 4 MICs (µM) 5 b c d e 1 2 3 4 5 6 7 8 Mel Surf PMB Van 6 Strains 7 Gram-negative bacteria 8 E. coli O157:H7 21530 >71.15 >70.09 16.75 7.94 3.67 6.97 0.85 3.32 0.35 >123.51 3.07 >88.32 9 >71.15 >70.09 >67.01 31.77 14.66 27.89 1.70 13.29 0.35 >123.51 0.38 88.32 10 E. coli 25922 >71.15 >70.09 >67.01 31.77 29.32 13.94 1.70 26.59 1.41 >123.51 1.54 >88.32 11 E. coli 35150 12 S. paratyphi-A 20501 >71.15 >70.09 33.50 15.89 7.33 3.49 0.43 6.65 0.70 >123.51 0.77 >88.32 13 >71.15 >70.09 >67.01 15.89 3.67 6.97 0.85 3.32 0.35 >123.51 1.54 88.32 14 S. paratyphi-B 21495 >71.15 >70.09 >67.01 15.89 7.33 3.49 0.43 6.65 0.70 >123.51 3.07 88.32 15 S. paratyphi-C 21512 16 S. flexneri 51571 >71.15 >70.09 >67.01 15.89 7.33 3.49 0.43 1.66 0.70 >123.51 0.77 88.32 17 >71.15 >70.09 33.50 15.89 14.66 13.94 1.70 13.29 0.70 >123.51 1.54 >88.32 18 S.choleraesuis 13312 S.typhimurium 51005 >71.15 >70.09 >67.01 >63.54 >58.64 13.94 6.81 >53.18 2.81 >123.51 0.77 >88.32 19 20 S. enteritidis 21527 >71.15 >70.09 >67.01 >63.54 >58.64 >55.78 6.81 >53.18 1.41 >123.51 1.54 44.16 21 13.94 1.70 6.65 1.41 >123.51 0.77 88.32 22 V.parahaemolyticus 21617 >71.15 >70.09 >67.01 >63.54 29.32 23 Gram-positive bacteria 24 S. aureaus 29213 >71.15 >70.09 >67.01 31.77 14.66 3.49 0.43 6.65 1.41 61.75 49.17 0.69 25 >71.15 70.09 >67.01 15.89 7.33 1.74 0.43 1.66 2.81 61.75 49.17 1.38 26 S. aureaus 1.160 15.89 7.33 1.74 0.21 3.32 0.18 123.51 24.59 0.69 27 L.monocytogenes 21662 >71.15 >70.09 33.50 28 L.monocytogenes 21633 >71.15 >70.09 67.01 31.77 7.33 1.74 0.21 3.32 0.35 123.51 12.29 0.69 29 >71.15 >70.09 >67.01 >63.54 14.66 55.78 0.85 53.18 1.41 123.51 49.17 0.17 30 B. cereus 63301 M. luteus 28001 >71.15 >70.09 >67.01 >63.54 7.33 27.89 0.43 13.29 0.70 123.51 >98.34 0.69 31 32 B. pumilus 63202 >71.15 >70.09 >67.01 >63.54 >58.64 55.78 0.85 26.59 0.35 61.75 49.17 0.35 33 >71.15 >70.09 >67.01 >63.54 29.32 55.78 0.43 53.18 1.41 123.51 49.17 0.35 34 P. fluorescens 21620 g >71.15 >70.09 >67.01 >63.54 14.66 6.97 0.85 3.32 1.41 123.51 >98.34 1.38 35 MRSA43300 36 Yeast 37 >71.15 >70.09 >67.01 >63.54 29.32 13.94 3.40 26.59 2.81 123.51 49.17 >88.32 38 C. albicans 10231 Ⅰ 142.30 140.18 86.22 29.83 15.62 10.83 1.32 11.00 0.79 247.02 1.19 113.64 39 GM (-) 40 GM (+)Ⅱ 142.30 129.79 106.37 58.83 14.66 9.48 0.46 9.04 0.82 98.03 53.11 0.59 41 h >71.15 >70.09 >67.01 >63.54 >58.64 >55.78 27.22 53.18 0.35 15.44 >98.34 >88.32 42 MHC Ⅰ i i 1.00 1.00 1.55 4.26 7.51 10.29 20.53 4.83 0.44 n.a. 164.62 n.a. TI (-) 43 i 44 TI (+)Ⅱ 1.00 1.08 1.26 2.16 8.00 11.76 59.15 5.88 0.43 0.16 298.16 n.a. 45 a b c d e f Minimum inhibitory concentrations. Melittin. Surfactin. Polymyxin B. Vancomycin. Amphotericin B. 46 47 g Methicillin-resistant S. aureus. Ⅰ GM (-) was the geometric mean of the peptide MICs against gram48 49 negative strains tested. Ⅱ GM (+) was the geometric mean of the peptide MICs against gram-positive 50 51 strains tested. hMHC was the minimum hemolytic concentration that induced 10% hemolysis of human red 52 53 Ⅱ blood cells. Ⅰ TI (-) was the therapeutic index that was calculated as MHC/GM (-). TI (+) was the 54 55 therapeutic index that was calculated as MHC/GM (+). iNot available due to little bioactivity. 56 57 58 59 60 ACS Paragon Plus Environment

f

AMB

>138.51 >138.51 >138.51 >138.51 >138.51 >138.51 >138.51 >138.51 >138.51 >138.51 >138.51

>138.51 >138.51 >138.51 >138.51 >138.51 >138.51 >138.51 >138.51 >138.51

4.33 277.02 277.02 0.54 n.a.

i i

n.a.


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 52

Table 4. The effects of physiological salts on the antibacterial activities of peptides. a

MICs (µM)

Peptides

Control

NaClb

KClb

NH4Clb

MgCl2b

CaCl2b

FeCl3b

Gram-negative strain E. coli O157:H7 1

>71.15

>71.15

>71.15

>71.15

>71.15

>71.15

>71.15

2

>70.09

>70.09

>70.09

>70.09

>70.09

>70.09

>70.09

3

16.75

>67.01

4.19

8.35

67.01

>67.01

>67.01

4

7.94

15.89

3.97

7.94

7.94

15.89

>63.54

5

3.67

14.66

3.67

3.67

3.67

7.33

>58.64

6

6.97

13.94

3.49

3.49

6.97

13.94

>55.78

7

0.85

3.40

0.85

0.85

0.85

3.40

6.81

8

3.32

>53.18

1.66

>53.18

>53.18

>53.18

>53.18

Gram-positive strain S. aureus 29213 1

>71.15

>71.15

>71.15

>71.15

>71.15

>71.15

>71.15

2

>70.09

>70.09

>70.09

>70.09

>70.09

>70.09

>70.09

3

>67.01

>67.01

>67.01

>67.01

>67.01

>67.01

>67.01

4

31.77

>63.54

>63.54

>63.54

>63.54

>63.54

>63.54

5

14.66

14.66

7.33

7.33

3.67

7.33

>58.64

6

3.49

6.97

0.87

1.74

1.74

3.49

>55.76

7

0.43

1.70

0.21

0.43

0.21

0.43

55.04

8

13.29

26.59

1.66

1.66

0.83

13.29

>53.18

a

b

Minimum inhibitory concentrations (MIC). The final concentrations of NaCl, KCl, NH4Cl, MgCl2, CaCl2 and

FeCl3 were 150 mM, 4.5 mM, 6 µM, 1 mM, 2 mM and 4 µM, respectively. The control MICs were measured without the presence of the salt ions.


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Table 5. Cell cycle distribution of the HepG2 cells treated with the engineered peptides. Peptide (µM)

HepG2 Sub-G1

G0/G1

S

G2/M

0.07

54.61

20.51

28.75

32

1.06

52.45

25.26

29.33

64

2.26

34.77

31.61

30.85

32

24.8

21.35

33.17

21.23

64

77.66

5.83

11.48

5.67

Control 1

7



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Figure 1. Helical wheel presentations of the engineered peptides. The yellow pointed to the hydrophobic residues. The blue pointed to the positively charged hydrophilic residues. The red pointed to the negatively charged hydrophilic residues. The purple pointed to non-charged polar residues. The gray represented other residues.


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Figure 2. Three-dimensional structure projections of Arg, Leu, 1 and 7. Red sticks represented positivelycharged polar amino acids. Green sticks represented nonpolar amino acids.



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Figure 3. Cytotoxicity of the designed peptides against RAW264.7 cells. Viability of RAW264.7 cells treated with the engineered peptides for 24 h was measured by MTT assay. Data shown were means ± SEM of three independent experiments.


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Figure 4. Flow cytometric analysis. Cell membrane disruption of E. coli O157:H7 cells treated with peptides 1 and 7 for 30 min was determined by an increase in fluorescent intensity of PI. (A) No peptide (negative control, 5.7%); (B) 1 (0.5 × MIC, 14.3%); (C) 1 (1 × MIC, 22.9%); (D) 1 (2 × MIC, 27.7 %); (E) 7 (0.5 × MIC, 78.9%); (F) 7 (1 × MIC, 91.1%); (G) 7 (2 × MIC, 98.0%).



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Figure 5. SEM micrographs of E. coli O157:H7 (up) and S. aureaus 29213 (down) cells treated with 1 and 7 at their 1 × MICs. The control was done without peptides.


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Figure 6. TEM micrographs of E. coli O157:H7 (up) and S. aureaus 29213 (down) cells treated with 1 and 7 at their 1 × MICs for 60 min. The control was done without peptides.



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Figure 7. Confocal fluorescence microscopic images of E. coli O157:H7 treated with FITC-labelled 1 and 7 at 1 × MIC for 30 min. Panels on the left, middle and right represented laser-scanning, merged and transmitted-light scanning images of bacterial cells, respectively. The control was done without peptides.


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Figure 8. Peptide binding affinity to LPS from E. coli 0111:B4 was measured using the BODIPY-TRcadaverine displacement assay. The fluorescence intensity was determined at an excitation wavelength of 580 nm and an emission wavelength of 620 nm. Data shown were means ± SEM of three independent experiments. AU, arbitrary units.



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Figure 9. Effects of the peptides on LPS-stimulated (A) NO and (B) TNF-α production in RAW264.7 cells. Amounts of TNF-α and NO in cell culture supernatants were determined by ELISA and Griess reagent, respectively. Data shown were means ± SEM of three independent experiments.


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Figure 10. Cytotoxicity of the designed peptides against HepG2 cells. The cells were incubated with various concentrations of peptides for 24 h, and the cell viability was determined by the MTT assay. Data shown were means ± SEM of three independent experiments.



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Figure 11. Morphological changes of HepG2 cells revealed by the inverted microscope (A, E) and SEM (B, C, D, F, G and H). A−D: Control HepG2 cells, without peptide treatment; E−H: 7 treated HepG2 cells, with peptide concentration fixed at 32 µM. A pore was indicated by the arrow.


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Figure 12. Induction of Sub-G1 arrest in HepG2 cells by the peptides. The cells were incubated with (A) No peptide (negative control); (B) 1 (32 µM); (C) 1 (64 µM); (D) 7 (32 µM) and (E) 7 (64 µM) for 24 h, and stained with PI. The DNA contents were measured using flow cytometry.



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Figure 13. Apoptosis of HepG2 cells treated with peptide 7 examined by DAPI staining. HepG2 cells were incubated with 32 µΜ peptide 7 for 0, 2, 4 and 8 h. Nuclei were visualized by DAPI (blue). The images were captured from confocal microscopy. The arrows pointed to the nucleus buds.


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Figure 14. ROS generation in peptide-treated HepG2 cells. (A) ROS generation in HepG2 cells after treatment with various concentrations of the engineered peptides for 8 hours was measured using DCFHDA. (B) DCFH fluorescence intensity in HepG2 cells treated with peptide 7 at 32 µM for 2-8 h was measured by LSCM.



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Table of Content Graphic (84 × 55 mm)


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Insights into the Antimicrobial Activity and Cytotoxicity of Engineered Î±

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