Synthesis and Mechanism Insight of a Peptide-Grafted Hyperbranched

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Synthesis and Mechanism Insight of a Peptide-Grafted Hyperbranched Polymer Nano-Sheet with Weak Positive Charges but Excellent Intrinsically Antibacterial Efficacy Jingyi Gao, Mingzhi Wang, Fangyingkai Wang, and Jianzhong Du Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b00307 • Publication Date (Web): 14 May 2016 Downloaded from http://pubs.acs.org on May 23, 2016

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Synthesis and Mechanism Insight of a PeptideGrafted Hyperbranched Polymer Nano-Sheet with Weak Positive Charges but Excellent Intrinsically Antibacterial Efficacy Jingyi Gao,†,‡,ǁ Mingzhi Wang, ‡,ǁ Fangyingkai Wang,‡ and Jianzhong Du†,‡ ,*

†

Shanghai Tenth People’s Hospital, Tongji University School of Medicine, 301 Middle

Yanchang Road, Shanghai 200072, China. ‡

Department of Polymeric Materials, School of Materials Science and Engineering, Key

Laboratory of Advanced Department Civil Engineering Materials of Ministry of Education, Tongji University, 4800 Caoan Road, Shanghai 201804, China.

ACS Paragon Plus Environment

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KEYWORDS Hyperbranched polymer; Self-assembly; Antibacterial efficacy; Nano-sheet; Antimicrobial resistance ABSTRACT: Antimicrobial resistance is an increasingly problematic issue in the world and there is a present and urgent need to develop new antimicrobial therapies without drug resistance. Antibacterial polymers are less susceptible to drug resistance but they are prone to inducing serious side effects due to high positive charge. Herein we report a peptide-grafted hyperbranched polymer which can self-assemble into unusual nano-sheets with highly effective intrinsically antibacterial activity but weak positive charges (+ 6.1 mV). The hyperbranched polymer was synthesized by sequential Michael addition-based thiol-ene and free radical mediated thiol-ene reactions, and followed by ring-opening polymerization of Ncarboxyanhydrides (NCAs). The nano-sheet structure was confirmed by transmission electron microscopy (TEM) and atomic force microscopy (AFM) studies. Furthermore, a novel “wrapping and penetrating” antibacterial mechanism of the nano-sheets was revealed by TEM and it is the key to significantly decrease the positive charges but have a very low minimum inhibitory concentration (MIC) of 16 µg mL-1 against typical Gram-positive and Gram-negative bacteria. Overall, our synthetic strategy demonstrates a new insight for synthesizing antibacterial nanomaterials with weak positive charges. Moreover, the unique antibacterial mechanism of our nano-sheets may be extended for designing next-generation antibacterial agents without drug resistance.

INTRODUCTION Antibiotics can inhibit or destroy bacteria or other microorganisms at low concentrations. Traditional antibiotics sterilize in chemical ways either by blocking the


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synthesis of bacteria cell walls, bacteria proteins or nucleic acids, or by strengthening the permeability of bacteria so that the bacteria will absorb much water and then burst, leading to drug resistance. The antimicrobial resistance is an increasingly problematic issue that leads to millions of deaths every year and bacterial antibiotic resistance poses one of the largest threat to public health. Clearly, there is a present and urgent need to develop new antimicrobial therapies.1-3 For example, intrinsically antibacterial polymeric materials have recently attracted much attention because they are less susceptible to development of resistance by bacteria.1-7 However, they are usually highly positively charged due to the requirement of strong electrostatic interaction between them and bacteria cell membrane,8,9 leading to side effects such as hemolysis and cytotoxicity toward human cells.10-12 Therefore, it is an important challenge to design intrinsically highly effective antibacterial materials with weak positive charges. Hyperbranched polymers are three-dimensional macromolecules13 whose structure can be tailored with diverse properties such as monodispersity14 and multivalency.15 They can also be self-assembled into a range of functional nanostructures such as vesicles.16,17 Furthermore, they may be used as drugs,18,19 drug carriers,20,21 and tissue engineering materials.20 On the other hand, antibacterial peptides can inhibit and kill a broad range of bacteria and fungi without antibiotic resistance.2,13,22 Moreover, the antibacterial efficacy of those linear antibacterial peptides can be significantly enhanced when self-assembled into polymer vesicles (a hollow polymeric nanoparticle) due to locally amplified concentration of positive charges of polymer vesicles.2,22


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Herein, we will design a highly effective antibacterial nano-sheet based on a multifunctional antibacterial peptide-grafted hyperbranched polymer to conquer the above mentioned challenges (Scheme 1). Furthermore, we also investigate the formation and the “wrapping and penetrating” antibacterial mechanism of nano-sheets (Scheme 2). Scheme 1. Synthesis of highly effective antibacterial peptide-grafted hyperbranched polymer.


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Scheme 2. Schematic illustration of the formation of intrinsically antibacterial nano-sheets by peptide-grafted hyperbranched polymer and subsequent “wrapping and penetrating” antibacterial mechanism. a)

a)

The antibacterial nano-sheet is self-assembled from peptide-grafted hyperbranched polymer 6.

Such thin nano-sheet can wrap the bacteria by electrostatic interactions and then kill them by subsequent membrane penetration.

EXPERIMENTAL SECTION Materials. D, L-Homocysteinethiolactone hydrochloride (Aldrich, ≥ 99%), 2-propynylamine (Aldrich,

96%)

and

dimethylolpropionic

acid

(DMPA;

Alfa

Aesar,

99%).

N-ε-

Benzyloxycarbonyl-L-lysine, L-phenylalanine, phosphotungstic acid, triphosgene and hydrogen bromide (30% in acetic acid) were purchased from Shanghai Hanhong Chemical Co., Ltd. Trifluoroacetic acid (TFA), diethyl ether, tetrahydrofuran (THF), N,N-dimethylformamide


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(DMF) phosphate buffer and NaOH were purchased from Aladdin. DMF and THF were dried by reflux for 1 day in the presence of calcium hydride and sodium strips, respectively. Gramnegative bacterium E. coli (ATCC35218) and Gram-positive bacterium S. areus (ATCC29213) were purchased from Nanjing bianzhen biological technology Co., Ltd. Other chemicals were used without further purification unless otherwise specified. Synthesis of Hyperbranched Polymers 3 and 4. A mixed solution of D, Lhomocysteinethiolactone hydrochloride (monomer 1; 4.00 g, 0.026 mol), 2-propynylamine (monomer 2; 1.5 g, 0.027 mol) and DMPA (0.34 g, 1.3 mmol) in DMF/H2O (24 mL, v/v = 2/1) was degassed via three freeze-thaw-pump cycles. After stirring for 4 h under UV light irradiation at room temperature, the reaction was terminated by exposure to air. Polymer 3 was precipitated from an excess of THF for three times. After drying overnight in a vacuum oven at room temperature, the product was obtained (3.00 g) with a yield of 60.8 %. The hydrochloride hyperbranched polymer 3 (1.6 g) and NaOH (0.31 g, 1 eq) were dissolved in water (3.0 mL) and the solution was stirred until pH 7. The resultant mixture was precipitated in an excess of diethyl ether one time. After drying overnight in a vacuum oven at room temperature, the amino-hyperbranched polymer 4 was obtained (1.35 g) with a yield of 84.4 %. Synthesis of Butyryl Chloride Terminated Hyperbranched Polymer (Polymer 7) for Calculating the Degree of Branching. In order to avoid the influence of the amino group when calculating the degree of branching, the amino groups in polymer 4 were reacted with the butyryl chloride. The amino-hyperbranched polymer 4 (100 mg), butyryl chloride (100 mg, 2 eq) and triethylamine (101 mg, 2 eq) were dissolved in DMF (1.0 mL). After stirring for 12 h at room temperature, the resultant product was precipitated in an excess of THF for twice and in diethyl


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ether for one time. After drying overnight in a vacuum oven at room temperature, polymer 7 was obtained. The corresponding 1H NMR spectra of polymers 4 and 7 were shown in Figure S1 and Figure S2 in the supporting information, respectively. The degree of branching (DB) was calculated according to the following equation.23,24 DB =

2I(dendrit ic) 2D = 2D + L 2I(dendrit ic) + I(linear)

Where D and L are the number of dendritic, and linear units, respectively and I denotes the integral area of protons. The DB of polymer 4 was calculated to be 93.3%.24 Synthesis of Antibacterial Peptide-Grafted Hyperbranched Polymers (Polymers 5 and 6). ZLys-NCA and Phe-NCA monomers were obtained according to the previously reported method.22 Then hyperbranched polymer 4 (0.40 g), Z-Lys-NCA (0.59 g) and Phe-NCA (0.37 g) were dissolved in DMF (15 mL) in a dried flask. The mixture was stirred at room temperature in a vacuum for 24 h. The purified polymer 5 was obtained by precipitation in water and freezedrying for 48 h (Yield: ~81%). Then polymer 5 was dissolved in excess HBr (15.0 mL, 30% in acetic acid) and stirred for 4 h, finally polymer 6 was obtained by precipitating in diethyl ether and subsequent dialysis against deionized water for 48 h (Yield: ~60%). The corresponding 1H NMR analyses were shown in Figures S4 and S5 in the Supporting Information, respectively. Self-Assembly of Polymer 6 into Nano-Sheets. Polymer 6 (9.0 mg) was dissolved in 3.0 mL of DMSO. A drop of TFA was added to break the hydrogen bonding. Then, 6.0 mL of deionized water was dropped into the solution by a gas-tight syringe with a vigorous stirring in 10 min. After another 12 h stirring, the solution was dialyzed against deionized water for 24 h to remove


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DMSO and TFA. Antibacterial Test of Nano-Sheets Self-Assembled from Polymer 6. Polymer 6 nano-sheet solution (30 mg mL-1) was diluted to different concentrations using saline. Then a mixture of 1.0 mL of nano-sheet solution and 20 µL of microorganism solution was cultivated in a conical flask at 37 °C. The optical densities of the microorganism solution were measured at 600 nm as a function of time. The antibacterial activities of the nano-sheets were also evaluated by the Luria– Bertani (LB) medium broth microdilution method. The bacterial colony was counted directly on the agarose plates. The control is only broth-containing bacterial cells. More details can be obtained in our recent articles.22,25 AFM Study of Nano-Sheets. AFM analysis was conducted to test the height of the wrinkled nano-sheets. A clean silicon wafer (1 × 1 cm2) was washed in the acetone in the presence of ultrasound for 10 min. Then 10 µL of diluted sample solution was dropped onto the silicon wafer and dried at room temperature overnight. TEM Studies on the Antibacterial Mechanism of the Wrinkled Nano-Sheets. The TEM analysis was conducted to reveal the antibacterial mechanism. The bacterial morphologies of both E. coli and S. aureus were viewed before and after the treatments with the wrinkled nano-sheets for 4 hours. The samples were prepared following a previously reported method.4,26 CMC. The critical concentration of the nano-sheet formation is defined as the lowest concentration of polymers to form nano-sheets in water. Pyrene was used as the probe to detect the formation of nano-sheets according to the previous protocol.22 Fluorescence intensities were recorded by exciting samples at 334 nm via a Lumina Fluorescence Spectrometer (Thermo Fisher).


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Cytotoxicity Study and Hemolysis Test. The cytotoxicity of polymer nano-sheets against normal liver cells (L02) was evaluated by using a sensitive colorimetric CCK-8 (Cell Counting Kit-8) assay. L02 was incubated with the polymer nano-sheets at various concentrations from 25 µg/mL to 150 µg/mL over 24 h. In the haemolysis test, the H50 value (i.e., the concentration causing haemolysis of 50% of red blood cells) was used to evaluate nano-sheet’s ability to lyse red blood cells. The lysis of 0.1% Triton X-100 was determined as positive control. Different concentrations of nano-sheets were treated with red blood cells (5%, v/v) in PBS for 1 h at 37 ℃. The absorbance value of supernatants was analyzed at 576 nm. More details can be obtained in our recent article.2 Characterization. GPC. The molecular weights and polydispersities of polymers 4 and 7 were determined by a gel permeation chromatography (GPC) performed at 35 oC with three linear Styragel columns and a Waters 2414 differential refractive index (RI) detector. DMF was utilized as eluent with a flow rate of 1.0 mL•min-1, and polystyrene was used as standard. DMF GPC analysis (refractive index detector) gave Mn = 3,094 Da and Mw/Mn = 1.11 for polymer 4, and Mn = 5,912 Da and Mw/Mn = 1.65 for polymer 7 (see Figure S3 in the Supporting Information). 1

H NMR. The NMR spectra were recorded using a Bruker AV 400 MHz spectrometer, with

DMSO-d6 or CDCl3 as solvent and TMS as standard at room temperature. In some cases CF3COOD was added to break the hydrogen bonding in polypeptides. TEM. TEM images were taken with a JEOL JEM-2100F instrument at 200 kV equipped with a Gatan 894 Ultrascan 1 k CCD camera. A drop of 3.0 µL of diluted nano-sheet solution was laid on the carbon-coated copper grid and dried at ambient environment. Then the bottom of the grid


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was turned upside on the phosphotungstic acid (PTA; 1%, pH 7) solution for 1 min. The excess phosphotungstic acid was drained off carefully through filter paper. After that, the grids were dried under ambient environment overnight. AFM. The AFM analysis was conducted on a Seiko (SPA-300 HV) instrument operating in tapping mode at 200-400 kHz drive frequency. Zeta Potential. The zeta potential study of nano-sheets in water was determined using NanoZS 90 Nanosizer (Malvern Instruments Ltd., Worcestershire, U.K.) at a fixed scattering angle of 90o. FTIR. Fourier transform infrared (FTIR) analysis of polymers were carried out on a Bruker EQUINOXSS/HYPERION2000 spectrometer equipped with OMNIC software in the frequency range of 500 - 4000 cm-1. XPS. X-ray photoelectron spectroscopy (XPS) measurement was performed by a PHI 5000C ESCA System XPS (Perkin Elmer Co., America) with aluminum target, high voltage of 14.0 kV and power of 300 W. RESULTS AND DISCUSSION Synthesis and Characterization of Antibacterial Peptide-Grafted Hyperbranched Polymers. We propose a new protocol to synthesize antibacterial peptide-grafted hyperbranched polymers (Scheme 1), which can be self-assembled into nano-sheets with highly effective intrinsically antibacterial efficacy (Scheme 2). The antibacterial hyperbranched polymer 6 was synthesized in four steps (Scheme 1). First, a hyperbranched polymer 3 was synthesized by sequential Michael addition-based


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thiol-ene and free radical mediated thiol-ene reactions.27,28 The hyperbranched structure was induced by the irradiation of UV light. Second, polymer 4 was obtained by adding NaOH solution to adjust pH to 7. Also, to calculate the degree of branching (DB) of hyperbranched polymers, the amine groups in polymer 4 were terminated by butyryl chloride to afford polymer 7 (Scheme S1 in the Supporting Information). Third, the ringopening polymerization of Z-Lys-NCA and Phe-NCA mononers afforded hyperbranched polymer 5 as antibacterial groups (here Lys and Phe stand for Lysine and phenylalanine, respectively). Overall, one amino group in the dendrite initiated the ring-opening polymerization of five Z-Lys and four Phe monomers. Finally, the deprotection of amino groups in polymer 5 afforded the final antibacterial peptide-grafted hyperbranched polymer 6. The peptide chains grafted on the hyperbranched polymer not only make polymer have more efficient antibacterial ability, but also contribute to self-assemble into complex nanostructures.29 The chemical structures of hyperbranched polymers were confirmed by 1H NMR spectra in DMSO-d6 (Figures S2, S4 and S5 in the Supporting Information). The molecular weights of polymers 4 and 7 were also evaluated by GPC (Figure S3 in the Supporting Information). The degree of branching of polymer 4 is calculated to be 93.3% according to the reported protocol (see the Supporting Information).23,24 Polymer 4 has a molecular weight over 3000 and very low polydispersity of 1.11. Analysis of the Membrane Thickness of the Wrinkled Nano-Sheets by TEM. The antibacterial peptide-grafted hyperbranched polymer 6 can form nano-sheets in water, as confirmed by transmission electron microscopy (TEM) study in Figure 1 and atomic force microscopy (AFM) study (Figure S6 in the Supporting Information).


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Figure 1. (A and B) TEM images of wrinkled nano-sheets; (C and D) Simulated electron transmittance of the red line in (B) and the relationship with the thickness (d) of the wrinkled sheets; (E) Histogram chart along the red scan line in (B). The TEM image is a function of electron density transmitting the nano-sheet. The vertical thickness (L) can be treated as a function of the membrane thickness (d) in the case of folded membrane of the wrinkled nano-sheet. The following equation is the analogue computation between the vertical thickness (L) and the membrane thickness (d).30


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The equation can be illustrated separately into two parts by the following simulation.

In the above equations, d stands for the membrane thickness; L represents the vertical thickness of membrane-folded wrinkled nano-sheet; R is the outer radii; x refers to distance along the diameter. From the histogram in Figure 1E, the length of 2d can be measured as 16.7 nm, thus the membrane thickness d can be calculated as ~8.4 nm. The area of wrinkled sheets is over 50 µm2. Formation Mechanism of Peptide-Grafted Nano-Sheets with Weak Positive Charge. The wrinkled nano-structure is very similar to our previously reported polymer vesicle membrane structure.31 According to the AFM result (Figure S6 in the Supporting Information), the corresponding height (ca. 3.78 nm) reveals the nano-sheet is rough and very thin, which is in agreement with the wrinkled nano-sheet structure in the TEM study (Figure 1).29,32 The formation of antibacterial nano-sheets may be related to the special structure of the hyperbranched polymer with superiority in tailoring the organization with molecular-level precision.33,34 Furthermore, it can provide multiple noncovalent interactions such as


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hydrogen bonding, π-π stacking, hydrophobic effect and electrostatic interactions for facilitating the nano-sheet formation. 35,36 Moreover, the peptide units’ strong interplay between hydrogen bonding, π-π interactions29,32 and rigid hydrophobic phenyl groups makes the self-assembled nanosheets very stable in water.35,37 Furthermore, these factors will result in larger bending energy than the edge energy, leading to the formation of nano-sheets.38,39 Polymer 3 can be dissolved in water but polymer 4 can’t be dissolved in water, indicating that there was strong hydrogen bonding in polymer 4 with amine groups. This suggests that the hydrogen bonding in polymer 6 is only weakened but not completely destroyed after reaction of polymer 4 with Z-Lys and Phe. From the N 1s photoemission line in the XPS spectra of nano-sheets (Figure 2A), the characteristic peaks of amine and amide groups with the peaks at 399.2 eV and 401.0 eV can be observed, indicating the possibility for the hydrogen bonding.40,41 Furthermore, in the ATR-FTIR spectra (Figure 2B), the peak at 3276 cm−1 indicates the presence of the N-H groups, while the bands with the maximums at 1629 cm-1(C=O stretching), and around 1534 cm-1 (C-N stretching and CO-N-H bending) correspond to motions associated with the amide group. The above results suggest the hydrogen bonding in the nano-sheets. The weak positive charge in the nano-sheets is caused by partial amine groups in polymer 6 solvated in water (whereas most of amine groups are not solvated due to hydrogen bonding in the nano-sheets).


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A

N1s C-N -NH2

12000

8000

Transmittance (%)

16000

Intensity (a.u.)

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B

1629

3276

1534

4000

394

396

398

400

402

Binding Energy (eV)

404

4000

3500

3000

2500

2000

1500

1000

-1

Wavenumber (cm )

Figure 2. XPS spectra (A) and FTIR-ATR spectra (B) of nano-sheets. Antibacterial Activity and Mechanism of Nano-Sheets. The nano-sheets are weakly positively charged in water with a Zeta potential value of + 6.1 mV. It is well known that bacteria are negatively charged. Therefore, the nano-sheets can drift in water and adhere to the bacteria by electrostatic interaction.2 Furthermore, the Phe units can penetrate the membrane of bacteria, killing bacteria without obvious resistance.42-44 To test this hypothesis, the dynamic antibacterial test was conducted to evaluate the antibacterial ability of the nano-sheets self-assembled from hyperbranched polymer 6. Unfortunately, polymer 4 didn't dissolve in water so that we were not able to evaluate its antibacterial activity for comparison. The solution containing both bacteria and nano-sheets was incubated for 24 h at 37 oC. The optical density of the solution was measured at 600 nm of wave-length at intervals. Figure 3 A and B revealed the antibacterial ability with a low MIC value (16.0 µg mL1

) against both Gram-negative E. coli (ATCC35218) and Gram-positive S. aureus (ATCC29213). Alternatively, Luria–Bertani (LB) medium broth microdilution method was used to

further confirm the antibacterial ability of nano-sheets. As shown in Figure 4, dense bacterial colonies were observed in the control group without any nano-sheet treatment,


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whereas only sporadic bacterial colonies could be observed in the presence of 16 µg mL−1 of nano-sheets. Figure 4 also revealed that 95.8% and 94.8% of E. coli and S. aureus were killed at 16 µg mL−1, further confirming the MIC value of 16 µg mL−1. The corresponding log reduction values were 1.38 and 1.29 for E. coli and S. aureus, respectively, as calculated according to the following equation:3,45,46 log ܴ݁݀‫ = ݊݋݅ݐܿݑ‬log‫ ܣ‬− log‫ܤ‬ Where A is the number of surviving colonies in the control and B is that in the nanosheet sample. Moreover, the lowest concentration of polymer 6 to form nano-sheets in water is 14.8 µg mL−1 (see Figure S7 in the Supporting Information). This value is lower than the MIC value (16 µg mL−1), which confirms that it is the nano-sheet not the individual polymer that inhibits bacteria growth in aqueous solution. Nano-sheet against E. coli

A

1.2 1.0 0.8 0.6

B

Control 8 µg/mL 16 µg/mL 32 µg/mL 64 µg/mL

1.6

OD at 600 nm

Control 8 µg/mL 16 µg/mL 32 µg/mL 64 µg/mL

1.4

Nano-sheet against S. aureus

1.8

1.6

OD at 600 nm

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1.4 1.2 1.0 0.8 0.6

0.4

0.4

0.2

0.2 0.0

0.0 0

2

4

6

8

10

12

24

0

2

4

6

8

10

12

24

Time (h)

Time (h)

Figure 3. Dose-dependent growth inhibition of bacteria in the presence of nano-sheets (Polymer 6) over 24 h.


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Figure 4. Digital photographs of LB agar in the absence (a and c) and in the presence (b and d) of antibacterial nano-sheets against E. coli (a and b) and S. aureus (c and d). The visible white dots are the bacterial colonies.


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Figure 5. TEM images of E. coli and S. aureus in the absence (A and B) and presence of (C and D) of antibacterial nano-sheets. (E and F) Magnified and color TEM images of (C and D). The red circles highlight the rupture of bacteria membranes after the treatment with the nano-sheets, indicating a membrane penetration antibacterial mechanism. Furthermore, TEM studies in Figure 5 confirms that it is the membrane penetration at multiple sites that causes the death of bacteria, as expected. Compared with our previous


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antibacterial polymer vesicles which are much smaller than bacteria,2,22 the antibacterial nano-sheets are bigger than bacteria, having more surface to contact and wrap bacteria than small vesicles. This “wrapping and penetrating” antibacterial process was confirmed by TEM study (Figure 5) and schematically illustrated in Scheme 2. In Figure 5, both Gram-positive bacteria and Gram-negative bacteria in the control group (A) and (B) don’t have any rupture in the absence of antibacterial nano-sheets. In a sharp contrast, as the red circles highlighted in (C) and (D), the Gram-negative E. coli and Gram-positive S. aureus were adhered to the surface of the antibacterial nano-sheets. The antibacterial peptides on the nano-sheets pierced the bacteria, destroyed the cell membrane and finally burst it, presenting excellent bactericidal ability. The Lys units in the nano-sheet can enhance the local charge concentration and promote the electrostatic interaction between the nano-sheets and the bacteria, whereas the Phe units with rigid benzene structure facilitate the cell penetration efficiency.15 Usually, the phenyl groups of the nano-sheets pierce into the cell membrane, mainly into the phospholipid part, so the bacteria contents flow out, causing the death of bacteria. Sometimes, as the concentration of the bacterial cytoplasm is much higher than the outer environment, the bacteria will swell by absorbing excess water, especially for Grampositive bacteria S. aureus. This unique “wrapping and penetrating” antibacterial mechanism attributes to the excellent antibacterial efficacy of the nano-sheets, even though these nano-sheets only have very weak positive charges. Cytotoxicity Study and Haemolysis Test of Nano-sheets. The cytotoxicity test against human normal liver cells (L02) was determined by CCK-8 over 24h (see Figure S8 in the


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Supporting Information). When the concentration of the nano-sheet solution was 50 µg mL-1, it showed low toxicity to cells. Also the red blood cell haemolysis experiment was performed to test the blood compatibility of nano-sheet (see Figure S9 in the Supporting Information). The H50 (i.e., the concentrations causing haemolysis of 50% of red blood cells) was around 400 µg mL-1, which was higher than the MIC (16 µg mL-1). Therefore, both cytotoxicity and haemolysis test revealed that the nano-sheets may be used as antibacterial agents.

CONCLUSIONS In summary, we have designed and synthesized an antibacterial peptide-grafted hyperbranched polymer. TEM and AFM studies confirmed that the hyperbranched polymer can self-assemble into nano-sheets. TEM studies also revealed that the nano-sheets can wrap the bacteria, penetrate the bacteria cell membranes and then kill the bacteria, giving a very low MICs against both Gram-negative E. coli and Gram-positive S. aureus (16 µg mL-1). Furthermore, the nano-sheets have very weak cationic charges (+ 6.1 mV) due to the hydrogen bonding of amine groups, which is much lower than most of antibacterial materials. Therefore, our strategy provides us with a new insight for synthesizing highly effective intrinsically antibacterial agents with low cationic charges in aqueous media for broad medical applications. Moreover, this unique ‘wrapping and penetrating’ antibacterial mechanism will strengthen the confidence of scientists to design new antibacterial materials without antibiotic resistance.


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ASSOCIATED CONTENT Supporting Information: Scheme S1, Figures S1–S9, Tables S1 for synthetic route, GPC, NMR, AFM, CMC, cytotoxicity, and haemolysis studies. This material is available free of charge via the Internet at http://pubs.acs.org online. Correspondence and requests for materials should be addressed to J. Du. AUTHOR INFORMATION * Corresponding author Email: [email protected]

Author Contributions ‖

These authors contributed equally.

ACKNOWLEDGMENT This work is supported by NSFC (21174107, 21374080 and 2151101151), Shanghai 1000 Plan (SH01068), Shanghai International Scientific Collaboration Fund (15230724500), the Fundamental Research Funds for the Central Universities (0500219211 and 1500219107) and the open fund of Beijing National Laboratory for Molecular Sciences (20140127). REFERENCES

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Synthesis and Mechanism Insight of a Peptide-Grafted Hyperbranched

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