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Preparation and Antibacterial Mechanism Insight of PolypeptideBased Micelles with Excellent Antibacterial Activities Yuejing Xi, Tao Song, Songyao Tang, Nuosha Wang, and Jianzhong Du Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b01285 • Publication Date (Web): 02 Nov 2016 Downloaded from http://pubs.acs.org on November 3, 2016

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Preparation and Antibacterial Mechanism Insight of Polypeptide-Based Micelles with Excellent Antibacterial Activities Yuejing Xia,b,§, Tao Songb,§, Songyao Tangb, Nuosha Wangb and Jianzhong Dua,b,*

a

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

Yanchang Road, Shanghai 200072, China b

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

University, 4800 Caoan Road, Shanghai 201804, China. Email: [email protected]; Fax: +8621-6958 0239; Tel: +86-21-6958 0239

KEYWORDS. Polypeptide; Micelles; Antibacterial; Self-assembly; Polymer

ABSTRACT. Traditional antibiotics usually sterilize in chemical ways which may lead to serious drug resistance. In contrast, peptide-based antibacterial materials are less susceptible to drug resistance. Herein we report the preparation of an antibacterial peptide-based copolymer micelle and the investigation of their membrane-penetration antibacterial mechanism by transmission

electron

microscopy

(TEM).

The

copolymer

is

poly(L-lactide)-block-

ACS Paragon Plus Environment

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poly(phenylalanine-stat-lysine) [PLLA31-b-poly(Phe24-stat-Lys36)] which is synthesized by ringopening polymerization. The PLLA chains form the core whereas the polypeptide chains form the coronas of the micelle in aqueous solution. This micelle boasts excellent antibacterial efficacy against both Gram-positive and Gram-negative bacteria. Furthermore, TEM studies clearly reveal that the micelles pierce and then destroy the cell membrane of the bacteria. We also compared the advantages and disadvantages of two general methods for measuring the Minimal Inhibitory Concentration (MIC) values of antibacterial micelles. Overall, this study provides us with direct evidence for the antibacterial mechanism of polypeptide-based micelles and a strategy for synthesizing biodegradable antibacterial nanomaterials without antibiotic resistance.

Introduction. Bacterial infection is a common disease in our daily life. It is usually treated by systemic or local administration, or intravenous injection of low-molecular-weight antibiotics.1-3 Traditional antibacterial mechanism can be divided into two types. One is to penetrate into the target microorganism and work on specific targets. For example, breaking double-stranded DNA through the inhibition of DNA gyrase, blocking cell division and interfering with peptidoglycan.2,4 The other is to induce bacterial lysis by interaction with the bacterial membrane and/or release of cell wall lytic enzymes, which leads to their spontaneous burst.5,6 Both of them are chemical sterilization ways, as most conventional antibiotics (such as ciprofloxacin, doxycycline and ceftazidime) can’t damage the cell wall physically.7,8 In this way, the morphology of bacteria is preserved and the bacteria can easily develop drug resistance. As a result, the antibiotics no longer work well in the near future, which makes the side effects even


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worse and accelerates the gene mutation and the development of word-wide problem of drug resistance.1 Fortunately, in nature, a small number of peptides have excellent antibacterial activities.7-12 Most antimicrobial peptides have positive charges and amphipathic features, and their antimicrobial activities largely depend on the formation of facially amphiphilic α-helical or βsheet-like tubular structures. Then the positively charged peptides interact with negatively charged cell membranes, followed by diffusion through the cell walls and insertion into the lipophilic domain of the cell membranes. It is noteworthy that the disintegration of cell membrane eventually leads to death of cell without the development of drug resistance.5 Compared to these conventional antibiotics, natural peptides with excellent antibacterial activities are ideal next-generation antibacterial agents with less side effects.7-12 However, current natural antibacterial peptides are usually generated from nature at a high cost, which can’t meet the huge clinical demand.13,14 Pioneering work in translating peptide designs into synthetic polymers laid the foundation for the synthetic antibacterial polypeptides, especially the technique in the controlled polymerization of N-carboxyanhydrides (NCAs),12,15-17 which makes that antibacterial polypeptides can be synthesized in a large scale and at a low cost.15,18-20 Although some synthetic polypeptides were designed, few of them can pass the clinical trials because of their high cytotoxicity.21,22 Recently, a range of nanostructures were prepared by macromolecular self-assembly, showing promising potential in nanomedicine and other fields.2329

For example, recently, we reported polypeptide-based vesicles with excellent antibacterial

activities, good biocompatibility, and good biodegradability.30,31 However, the actual antibacterial mechanism of polymeric nanostructures such as vesicles and micelles remains an important challenge, which deserves to be revealed with direct evidence.3,32


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Herein, we design a new polypeptide-based micelle (see Scheme 1) to clearly reveal the antibacterial mechanism by TEM. Scheme 1. Illustration of Self-Assembly of Polypeptide-Based Copolymers into Micelles.

EXPERIMENTAL SECTION Materials. N-ε-benzyloxycarbonyl-L-lysine, L-phenylalanine, triphosgene and hydrogen bromide (30% in acetic acid) were purchased from Shanghai Hanhong Chemical Co., Ltd. Llactide was purchased from (Jinan Daigang Biomaterial Company). Hexane, stannous 2ethylhexanoate (Sn(Oct)2), trifluoroacetic acid (TFA), diethyl ether, tetrahydrofuran (THF), N,Ndimethylformamide (DMF), N-(tert-butoxycarbonyl)-2-aminoethanol, dichloromethane and methanol were purchased from Aladdin. DMF and THF were dried by reflux for 1 day in the presence of calcium hydride and sodium strips, respectively. Gram-negative bacterium E. coli (ATCC35218) and Gram-positive bacterium S. aureus (ATCC29213) were purchased from Nanjing Bianzhen Biological Technology Co., Ltd. Other chemicals were used without further purification unless otherwise specified. Synthesis of Z-Lys-NCA Monomer. N-ε-benzyloxycarbonyl-L-lysine (5.000 g, 17.84 mmol) and α-pinene (12.14 g, 89.19 mmol) were dissolved in 100 mL of anhydrous THF in a three-neck


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round-bottom flask with a magnetic flea. The triphosgene (3.979 g, 13.40 mmol) was dissolved in 30.0 mL of anhydrous THF in a constant pressure funnel. After a magnetic flea was placed into the three-neck round-bottom flask, the whole set of device was assembled and argon was blown in to exhaust the air inside. After heating to 50 oC, the triphosgene solution was added dropwise into the THF solution over a period of one hour under argon. The mixture would turn gradually clear in 4 hours. Then the reaction was allowed to cool to room temperature. The mixture was precipitated into 500.0 mL of hexane with fast stirring for three times. The obtained product could be used after 48 hours of freeze-drying. The 1H NMR spectrum was shown in Figure S1 in the Supporting Information. Yield: ~79%. Synthesis of Phe-NCA Monomer. L-phenylalanine (5.000 g, 30.26 mmol) and α-pinene (20.58 g, 151.4 mmol) were dissolved in 100.0 mL of anhydrous THF in a three-neck round-bottom flask with a magnetic flea. The triphosgene (6.745 g, 22.73 mmol) was dissolved in 30.0 mL of anhydrous THF in a constant pressure funnel. The rest of procedure is the same as the synthesis of Z-Lys-NCA monomer. The obtained product could be used after 48 hours of freeze-drying. The 1H NMR spectrum was shown in Figure S2 in the Supporting Information. Yield: ~83%. Synthesis of PLLA31-NH-Boc (polymer 3). A round-bottom flask with a magnetic flea was prepared and loaded with L-lactide (12.6 g, 175.0 mmol) and N-(tert-butoxycarbonyl)-2aminoethanol (0.7463 g, 4.630 mmol).The mixture was degassed with argon sparge for 35 mins. Then a drop of Sn(Oct)2 catalyst was added into the flask quickly with the protection of argon flow. After heating in an oil bath at 130 oC, the solid mixture melted to liquid gradually. The reaction solution was stirred for 36 hours at 130 oC under argon, and the product was allowed to cool down to room temperature. Then the product was dissolved into the anhydrous dichloromethane and poured dropwise into 500 mL of methanol under vigorous stirring. The


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purification process was repeated three times. Finally, the precipitate was harvested and dried in vacuum at 35 oC. The 1H NMR spectrum was shown in Figure S3 in the Supporting Information. Yield: ~88%. Synthesis of PLLA31-NH2 (polymer 4). PLLA31-NH-Boc (6.00 g) was dissolved in 15 mL of anhydrous dichloromethane under argon, and then 10 mL of anhydrous TFA was added. The reaction solution was stirring at room temperature for 2 hours. Then the reaction solution was poured dropwise in 300 mL of hexane under vigorous stirring. After the purification process, the solvents were removed under vacuum. Then the product was dissolved in anhydrous DMF again. The re-dissolved solution was transferred into a dialysis tube and dialyzed against 5% NaHCO3 aqueous solution and deionized water for 2 days to remove trace of residual TFA solution. Finally the solvents were removed under vacuum. The white solid product was dried in vacuum at 37 oC. The 1H NMR spectrum was shown in Figure S4 in the Supporting Information. Yield: ~75%. Synthesis of PLLA31-b-Poly[phenylalanine-stat-(Z-lysine)] (polymer 5). Z-Lys-NCA (1.00 g, 3.270 mmol) and Phe-NCA (0.4183 g, 2.190 mmol) were dissolved in anhydrous DMF in a round-bottom flask with a magnetic flea. Then PLLA-NH2 (0.25 g, 1.093 mmol) was added into the reaction solution. The mixture was stirred at room temperature in vacuum for 24 hours, and then the solvents were removed under vacuum. Excess water was added into the reaction flask to remove the residual DMF and this process was repeated for eight times. Then the crude polymer 5 was obtained by purification in water. Finally, the purified polymer 5 was obtained by freezedrying for 48 hours. The 1H NMR spectrum was shown in Figure S5 in the Supporting Information. Yield: ~67%.


6

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Synthesis of PLLA31-b-Poly(phenylalanine24-stat-lysine36) (polymer 6). The mixture of PLLA31-b-poly[Phe24-stat-(Z-Lys)36] was placed in a round-bottom flask with a magnetic flea. Excess HBr (15.0 mL, 30% in acetic acid) was added into the reaction flask. The reaction solution was stirred at room temperature for 5 hours. Then the solution was poured dropwise into 300 mL of anhydrous diethyl ether for 5 times. After purification, the crude product was dried under vacuum and then dissolved again in deionized water with two drops of TFA. The aqueous solution containing the desired copolymer was transferred into a dialysis tube, and dialyzed against 5% NaHCO3 aqueous solution and deionized water for 2 days to remove traces of HBr/CH3COOH solution and TFA. Finally, the purified polymer 6 was obtained by freezedrying for 48 hours. The 1H NMR spectrum was shown in Figure S6 in the Supporting Information. Yield: ~64%. Self-assembling Polymer into Micelles. PLLA31-b-poly(Phe24-stat-Lys36) (10.0 mg) was dissolved in 3.0 mL of anhydrous DMSO. One drop of TFA was added to break the hydrogen bonding to ensure the complete dissolution of the copolymer. Then, 6.0 mL of deionized water was added dropwise into the solution by a buret with vigorous stirring for 10 mins. After stirring for another 2 hours, the solution was transferred into a dialyzed tube and dialyzed against deionized water for 48 hours to remove DMSO and TFA. The dialysis medium was changed for eight times in this process. In vitro Enzymatic Degradation. The micelles with a PLLA core can be degraded in the presence of the lipase. The degradation degree can be evaluated by the count rates of the micelle solution via Dynamic Light Scattering (DLS). The aqueous lipase solution (0.050 mL, 0.100 mL and 0.200 mL; 1000 µg mL-1) and 1.50 mL of micelles solution (400 µg mL-1) were added into deionized water (0.45 mL, 0.40 mL, 0.30 mL) to keep the concentration of all the micelles at


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300 µg mL-1, and the concentrations of lipase in different enzymatic degradation experiments were respectively 25, 50 and 100 µg mL-1, respectively. The mixture solution was in situ conducted in the DLS sample cuvettes at 37 oC. The micelle solution with lipase was degraded on a shaking bed at 37 oC. Then the derived count rates of the micelle solutions were monitored by DLS at different time intervals. Antibacterial Test. The broth microdilution method was used to determine the proper concentration range of the antibacterial polypeptide-based micelles. The Gram-positive bacteria (S. aureus) and the Gram-negative bacteria (E. coli) were cultivated in an oven at 37 oC. After this bacterial activation process, the Gram-positive bacteria (S. aureus, 5 × 107 cfu/mL) in the LB broth (0.10 mL) were placed on the 96-well culture plate. On the other hand, the antibacterial micelle solution was diluted with the LB broth to a range of concentrations (2000 to 16 µg mL-1). Then the diluted micelle solution (0.10 mL) was mixed with the bacteria in the LB broth on the 96-well culture plate. The bacteria in the presence of 1000 to 8.0 µg mL-1 of antibacterial micelles on the 96-well plate were cultured in a shaking bed at 100 rpm and 37 oC for 24 hours and the growth of the bacteria colony can be clearly distinguished by eyes. Judging from the growth of the bacteria colony, the proper concentration range of the antibacterial micelles was determined. The same procedures were repeated for Gram-negative bacteria (E. coli, 3 × 107 cfu/mL after activation) to determine the proper concentration range of the antibacterial micelles. Then the following two different methods were used to measure the MICs of the micelles.32 Method One was used to determine the MIC50 and MIC90 values of the polypeptide-based micelles against bacteria. (1) The sterilized LB broth (20 mL) was added in the sterilized culture dish. (2) Broth-containing bacteria (20 µL) which were cultured in the oven at 37 oC overnight


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were added into the above-mentioned sterilized LB broth (20 mL) and mixed up evenly to afford the diluted broth-containing bacteria suspension (for E. coli, 3 × 104 cfu/mL; for S. aureus, 5 × 104 cfu/mL). (3) The diluted broth-containing bacteria suspension (1.8 mL) was added in five 3.5 mL of sterilized quartz cuvettes. (4) Several concentrations of micelle solutions (0.2, 0.3, 0.4, 0.5, 0.6, 0.8, 1.0, 1.2 mg mL-1) were prepared by diluting the micelle solution (1.2 mg mL-1) with water. (5) Each micelle solution (200 µL) was added to each quartz cuvette to obtain the mixture of bacteria and micelles (the final concentrations of micelles were ranging from 20 µg mL-1 to 120 µg mL-1). (6) The bacteria in the cuvettes were cultivated in the shaking bed at 100 rpm and 37 oC for 24 h. (7) The optical densities of bacteria suspensions were measured at 600 nm wavelength every 2 hours using a UV-Vis spectroscopy. 200 µL of water was added to 1.8 mL of diluted broth-containing bacteria suspension, which was set as the control.30-32 Alternatively, Method Two was used to determine the MIC90, MIC99 and MIC99.99 values. (1) Several concentrations of polypeptide-based micelles solutions (0.3, 0.4, 0.45, 0.6, 0.8, 1.0 mg mL-1) were prepared by diluting the micelle solution with PBS buffer solution at 1.2 mg mL-1, 37 o

C and pH 7.4. (2) Each micelle solution (100 µL) was spread on the surface of the glass sheets.

The water was evaporated slowly at 37 oC to afford a micelle-coated glass sheet. (3) 10 µL of bacteria which were cultivated overnight in the LB broth (for E. coli, 3 × 107 cfu/mL; for S. aureus, 5 × 107 cfu/mL) were spread on the micelle-coated glass sheets and cultivated at 37 oC for 2 hours. (4) PBS (2 mL) was used to wash the bacteria suspension on the glass sheets. (5) 100 µL of bacteria suspension from the glass sheets was added in 900 µL of PBS in a 1.5 mL of centrifuge tube. (6) 100 µL of bacteria suspension in the centrifuge tube was added in 900 µL of PBS in another centrifuge tube. This dilution process was further repeated for 3 times (by 10-fold dilution). (7) 100 µL of each suspension was added in LB agar (10 mL) in the culture plate and


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was incubated for 2 days at 37 oC. (8) The colony forming units were counted. The bacteria at each concentration were incubated on three culture plates. The control group followed the above procedures but without micelles added. Determination of Critical Micellization Concentration (CMC). The CMC is defined as the lowest concentration of polymer 6 to form micelles in water. Pyrene (3.0 mg, 15 µmol), which was used as a probe to monitor the formation of micelles, was dissolved in acetone (25 mL). The pyrene solution (10 µL) was respectively added into eight centrifuge tubes. Then the acetone in tubes evaporated overnight. The micelle solution was diluted with deionized water into different concentrations, and 4.0 mL of solution at each concentration was extracted and transferred into the centrifuge tubes. After stirred for 8 hours, the intensity of solutions with different concentration of micelles and the same concentration of pyrene was evaluated by a Lumina fluorescence spectrometer. Fluorescence intensities of solutions were recorded by exciting samples at 334 nm. In this process, a 10 nm slit width for excitation and a 10 nm slit width for emission were utilized. The samples were scanned with an emission wavelength from 350 nm to 500 nm. The intensities of the I1 (371.7 nm) was chosen as the vibronic bands. The intensity values were plotted against the log of the concentration of each micelle sample. Finally, the first four intensities were processed by linear fit and the last four intensities were processed by another linear fit. The two lines intersected at one point. According the equations of two lines, the abscissa value (the log of concentration) of the point could be calculated, corresponding to the CMC of 13.2 µg mL-1. Cytotoxicity Study. Cell Counting Kit-8 (CCK-8) allows sensitive colorimetric assays to evaluate the number of viable cells in cell proliferation and cytotoxicity assays against normal liver cells (L02). The amount of the formazan dye which is a yellow-colored reduction product


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transformed from WST-8 due to the dehydrogenase activities can illustrate the cellular activities of liver cells. First, the L02 cells were cultivated in the each well of 96-well plates with the same density (4000 cells/well) in 100.0 µL of Dulbecco’s Modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) for 12 h, 24 h at 37 oC in a humidified 5% CO2-containing atmosphere. Second, 20.0 µL of solutions containing 25, 37.5, 50, 75, and 100 µg mL-1 of polymer 6 micelles were added and incubated for 24 h, respectively. The control group is untreated cells. Last, each well was added in CCK-8 dye and the plates were incubated for another 1 h at 37 oC, then the absorbance was measured by a microplate reader using dual wave length spectrophotometry at 450 nm and 630 nm. Every treatment was repeated five times. The relative cell viability (%) was determined by comparing the absorbance at 450 nm with control wells containing only cell culture medium. Characterization 1

H NMR Spectra Proton Nuclear Magnetic Resonance (1H NMR). The NMR spectra were

recorded using a Bruker AV 400MHz spectrometer, with DMSO-d6, or CDCl3 as solvent and TMS as standard at room temperature. For polymers containing polypeptide after polymerization, two drops of CD3COOD could be added to break the hydrogen bond. DLS Studies. The DLS studies of aqueous polypeptide-based micelles were conducted for three times using Nano-ZS 90 Nanosizer (Malvern Instruments Ltd., Worcestershire, UK) at a fixed scattering angle of 90°. The aqueous micelle solution was analyzed by disposable cuvettes. The data were processed by cumulative analysis of the experimental correlation function. The diameters of the micelles were calculated from the computed diffusion coefficients using the Stokes-Einstein equation.


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Scanning Electron Microscopy (SEM). SEM images were utilized to observe the surface morphologies of micelle structure. To obtain SEM images, a drop of solution was spread on a silicon wafer and left overnight to dry. Samples were coated with gold and viewed by a FEI Nova NanoSEM 450 electron microscopy operated at 500 V. Transmission Electron Microscopy (TEM). TEM images were taken with a JEOL JEM-2100F instrument at 200 kV equipped with a Gatan 894 Ultrascan 1 k CCD camera. To prepare TEM samples, 5 µL of diluted micelle solution was dropped onto a carbon-coated copper grid and dried in ambient environment. Phosphotungstic acid (PTA; 1%, pH 7.0) solution was dropped onto a hydrophobic film (parafilm), and then the sample-loaded grids were laid upside down on the top of the PTA solution for 1 min. The excess PTA solution was blotted up through a filter slightly. After that, the grids were dried under ambient environment overnight. TEM Study of Antibacterial Mechanism. The samples of bacteria in the presence (experiment group) and absence (control group) of antibacterial micelles for TEM study were prepared according to the following protocol. (1) Activation of bacteria. The bacteria were cultivated in the LB broth overnight in an oven at 37 oC. (2) Bacterial strain extraction. The activated bacteria in the LB broth (2.0 mL) were centrifuged at 5000 rpm (1500 g) for 20 mins and the supernatant was removed. Then additional 2.0 mL of broth-containing bacteria were added into this centrifuge tube again and the centrifuge/removal processes were repeated. These addition/centrifugation/removal processes were repeated once again. Finally, 6.0 mL of bacterial strain was extracted. (3) Purification of bacteria. 2.0 mL of normal saline was poured into the centrifuge tube. The residual supernatant was washed out after shaking the tube, centrifuging for 30 min and removing the supernatant. Then 2.0 mL of normal saline was added in the centrifuge tube again. Shake well and then divide the bacteria into two groups: experiment group (1.0 mL)


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and control group (1.0 mL). (4) Experiment group. The polymer micelle solution (0.05 mL; 400 µg mL-1) was added into the centrifuge tube with 1.0 mL of bacteria. The mixture of bacteria and micelles was shaken well and then cultivated in a shaking bed at 37 oC for 4 hours, followed by the centrifugation at 5000 rpm (1500 g) for 30 mins. The supernatant was removed and 2.0 mL of PBS was poured into the centrifuge tube. The residual supernatant was then removed after shaking the tube and centrifuging for 30 mins. (5) Control group. The bacteria (1.0 mL) in another centrifuge tube without the addition of antibacterial micelles were shaken well and then cultivated in a shaking bed at 37 oC for 4 hours, followed by centrifugation at 5000 rpm (1500 g) for 30 mins. The supernatant was removed and 2.0 mL of PBS was poured into the centrifuge tube. The tube was shaken well and centrifuged for 30 mins, then the residual supernatant was removed. (6) Immobilization of bacterial morphology. Phosphate buffer (pH 7.4; 0.5 mL) containing 2.5% glutaraldehyde was added to two centrifuge tubes of experiment and control groups. The tubes were shaken well and then placed in the fridge overnight at 4 oC to fix the bacterial morphology. (7) TEM sample preparation. The above two tubes were centrifuged for 30 mins and the supernatants were removed. Then the sterile water was added into both tubes. The tubes were shaken well and centrifuged for 30 mins. After the removal of the super supernatant, ca. 0.5 mL of sterile water was added into both tubes. These bacterial suspensions were then shaken well and ready for the preparation of samples for TEM observation (see TEM sample preparation section for details).5 UV-vis Spectroscopy. UV-vis studies were conducted using a UV-vis spectrophotometer (UV759S, Q/YXL270, Shanghai Precision & Scientific Instrument Co., Ltd) with a scan speed of 300 nm min-1. The absorbance and transmittance spectra of the micelles were recorded in the range of 600 nm.


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Fluorescence Spectroscopy. Fluorescence intensities of pyrene were measured with a fluorescence spectrometer (excitation at 334 nm and emission at 371.7 nm) via a Lumina Fluorescence Spectrometer (Thermo Fisher). GPC. A Waters Breeze 1525 GPC analysis system with two PL mix-D columns and a light scattering detector was utilized to perform Gel permeation chromatography (GPC) analysis. THF was used as eluent at a flow rate of 1.0 mL at 25 oC. Zeta Potential. The Zeta potential study of micelles in water was determined using Nano-ZS 90 Nanosizer (Malvern Instruments Ltd., Worcestershire, U.K.) at a fixed scattering angle of 90°. RESULTS AND DISCUSSION Synthesis and Characterization of Antibacterial Polypeptide-Based Polymers. The block copolymer, poly(L-lactide)-block-poly(phenylalanine-stat-lysine) [PLLA31-b-poly(Phe24-statLys36), polymer 6], was synthesized by ring-opening polymerizations of L-lactide and

N-

carboxyanhydrides (NCAs) of lysine and phenylalanine monomers,18,33 as shown in Scheme S1 in the Supporting Information. First, NCA monomers Z-Lys-NCA (Monomer 1 in Scheme S1; 1

H NMR spectrum in Figure S1 in the Supporting Information) and Phe-NCA (Monomer 2 in

Scheme S1; Figure S2) were synthesized by NCA ring forming reaction. Second, lactide was polymerized with N-(tert-butoxycarbonyl)-2-aminoethanol as the initiator to afford PLLA31-NHBoc (Polymer 3, Figure S3). Third, after the deprotection of PLLA31-NH-Boc and purification, PLLA31-NH2 (Polymer 4, Figure S4) was obtained. Z-Lys-NCA and Phe-NCA were mixed up with a DMF solution of PLLA31-NH2 for ring-opening polymerization to afford poly(L-lactide)block-poly[phenylalanine-stat-(Z-lysine)] [PLLA31-b-poly[Phe24-stat-(Z-Lys36)], Polymer 5, Figure S5]. Fourth, after the deprotection of lysine units and purification, the targeted poly(L-


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lactide)-block-poly(phenylalanine-stat-lysine) [PLLA31-b-poly(Phe24-stat-Lys36)] (Polymer 6, Figure S6) was obtained. The 1H NMR spectra and GPC analyses of the key monomers and copolymers were presented and discussed in the Supporting Information (Figures S1-S7). Analysis of the Morphology and Diameter of Antibacterial Micelles by SEM and TEM. We self-assembled PLLA31-b-poly(Phe24-stat-Lys36) (polymer 6) copolymer into micelles to investigate their antibacterial activity and antibacterial mechanism. The micelles were formed by polymer 6 in DMSO/H2O (1:2, v/v). Then the micelle solution was transferred in a dialysis tube in deionized water to remove DMSO. Finally the PLLA block forms the core of micelle and the antibacterial polypeptide block forms the corona.

Figure 1. (A) SEM and (B-D) TEM analysis of polypeptide-based micelles; (C) The zoom-in of (B); (D) The blue and green areas highlight the PLLA core of micelles. TEM samples were stained by 1% phosphotungstic acid.


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Scanning electron microscopy (SEM) studies confirmed the micellar structure with a numberaveraged diameter of 70 ± 6 nm (Figure 1A and Figure S10 in the Supporting Information). Dynamic light scattering (DLS) studies indicate an intensity-averaged hydrodynamic diameter (Dh) of 124 nm with a polydispersity index (PDI) of 0.271 (Figure S8 in the Supporting Information). Transmission electron microscopy (TEM) studies revealed the PLLA core of micelles with a number-averaged diameter of 25.5 ± 8 nm (Figure 1B-D and Figure S9 in the Supporting Information). The diameter shown in the TEM images is smaller than that determined by SEM and DLS studies because TEM reveals the PLLA core since it was stained by phosphotungstic acid34-36 whereas SEM and DLS studies presented the whole micellar structure. The phosphotungstic acid is also able to complex with antibacterial coronas through cation-anion interaction. However, it is usually not visible because the coronas are not densely aggregated. 3436

Antibacterial Activity of Micelles. In principle, the amino group of lysine (Lys) forms the amino cation in aqueous solution at physiological pH to possess positive charges, and then it can stick to the bacteria membrane via electrostatic interaction. Once the micelles are adhered to the surface of the bacteria, the antibacterial polypeptide changes the permeability of the bacteria and the phenyl group of phenylalanine may penetrate the bacteria.30,31 As to Gram-positive bacteria, the micelles can directly cross the peptidoglycan layer and result in disruption of the inner lipid membrane. As to Gram-negative bacteria, their surface consists of an outer membrane and an inner membrane with peptidoglycan between them. The micelles are firstly adsorbed to the outer membrane by electrostatic interactions, followed by the mild disruption of the outer membrane. Afterwards, the micelles diffuse through the outer membrane and the peptidoglycan to disrupt


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the inner membrane.37 In some cases, the damage to the membrane could change the permeability and inhibit bacterial respiration.7,38,39 In reality, the polypeptide-based micelles showed excellent antibacterial activity. MIC is used to evaluate the antibacterial effect of micelles against bacteria. Gram-negative E. coli (ATCC35218) and Gram-positive S. aureus (ATCC29213) were selected in the antibacterial tests. MIC50, MIC90, MIC99 and MIC99.99 are the lowest concentrations of micelles required to inhibit 50%, 90%, 99% and 99.99% bacteria growth, respectively.40,41 The MIC50 or MIC90 values were obtained by Method One based on the optical densities of the suspension containing the micelles, bacteria and LB broth in a quartz cuvette against the control group that contained bacteria and LB broth only. The growth rates of E. coli and S. aureus were shown in Figure 2. Furthermore, the MIC90, MIC99 and MIC99.99 values were obtained by Method Two for comparing the advantages and disadvantages of different methods for determining MIC90. The colony forming units were counted by naked eyes on the surface of the culture dish (Figure S12 in the Supporting Information) and the corresponding antibacterial rate and log reduction were provided in Table S1 in the Supporting Information.


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1.6 (B) Polymer micelles against S. aureus

1.6 (A) Polymer micelles against E. coli Control -1 20 µg mL -1 30 µg mL -1 40 µg mL -1 50 µg mL -1 60 µg mL

OD at 600 nm

1.2 1.0 0.8

1.2 1.0 0.8

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20 30 40 50 -160 Concentration of micelles (µg mL )

0.0

20 30 40 50 -160 Concentration of micelles (µg mL )

Figure 2. (A-B) Dose-dependent growth inhibition of bacteria in the presence of micelles made from polymer 6. (C-D) Absorbance at different concentrations for three typical observation times. OD: optical density.

The black column is the control group with the broth-containing bacteria only. The MIC50 and MIC90 values of polymer 6 micelles against both Gram-negative E. coli and Gram-positive S. aureus were 40 µg mL-1 versus the whole micelles (17.7 µg mL-1 versus lysine, Figure 2), and 120 µg mL-1 versus the whole micelles (53.1 µg mL-1 versus lysine, Figure S11 in the Supporting Information), respectively.


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Furthermore, the MIC90, MIC99 and MIC99.99 values of polymer 6 micelles against E. coli and S. aureus were determined by Method Two. The MIC values were 300 µg mL-1, 400 µg mL-1 and 600 µg mL-1 versus the whole micelles, respectively (Figures S11, S12 and Table S1 in the Supporting Information). However, this method may underestimate the antibacterial efficacy of polypeptide-based micelles. For example, the MIC90 value determined by this method is 300 µg mL-1, which is higher than that obtained from Method One by measuring the optical density of bacteria in the presence of micelles (120 µg mL-1). The possible reason is that for Method Two, the surface areas of the micelles decreases due to the aggregation of the micelles as water evaporates from the culture plate, leading to a “worse” antibacterial efficiency than that obtained from Method One. In contrast, for Method One, the suspension of micelles and bacteria are well dispersed in the LB broth in the cuvette, resulting in more contact surfaces between micelles and bacteria. However, the remaining oxygen in the cuvette (for cultivating bacteria and measuring the optical density) is less than that on the culture plates (Method Two). Therefore, the bacteria growth may be inhibited to some extent in the cuvette due to the lack of oxygen, leading to a “better” MIC value than that obtained from Method Two. Overall, both methods for determining the MIC values have their advantages and disadvantages. The real MIC values should be between the values determined by both methods. Antibacterial Mechanism of Antibacterial Micelles by TEM. TEM studies were further performed to reveal the antibacterial mechanism of polymer 6 micelles, as shown in Figure 3.


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Figure 3. Comparative TEM images of E. coli (A-D) and S. aureus (E-H) in the absence (A and E) and presence (B, C, D, F, G and H) of antibacterial micelles from polymer 6. The micelles which adhered to E. coli 1 surface and S. aureus 7 surface were highlighted by the yellow circles in TEM images (B) and (F). The rupture of E. coli 2-6 was highlighted by the red circles in TEM images (C-D) and the rupture of S. aureus 8-11 was highlighted by the red circles in TEM images (G-H). All the samples are stained by 1% phosphotungstic acid.

Both Gram-positive bacteria and Gram-negative bacteria in the control group (A and E) don’t have any rupture. As the yellow circles highlighted in (B) and (F), the antibacterial micelles adhered to the surface of the Gram-negative E. coli and Gram-positive S. aureus. In a sharp contrast, as the red circles highlighted in C, D, G and H, the Gram-negative E. coli in the presence of antibacterial micelles (C and D) and the Gram-positive S. aureus in the presence of antibacterial micelles (G and H) were ruptured as the micelles pierced the bacteria and destroyed the cell membrane and finally burst it, presenting excellent bacteria-killing ability. This is reasonable because the amino groups of the micelle coronas have positive charges and the


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bacteria are negatively charged, leading to electrostatic interaction between bacteria and the micelles. Moreover, the antibacterial polypeptide changes the permeability of bacteria through the membrane integration mechanism37 and the phenyl groups can even pierce into the bacteria, 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 Gram-positive bacteria S. aureus. TEM images revealed that the antibacterial mechanism of the micelles involves the adhesion and the rupture processes. It is noteworthy that during this experiment, as most of the bacteria are dead and the content of them outflow, the TEM sample must be washed with PBS and then sterile water to clean out the content (including some micelles) so that the visual field is clear. Also, some micelles can be degraded by enzymes released from bacteria. Thus most of the micelles were not visible in the TEM image. More comparative colored TEM images of E. coli (A-C) and S. aureus (D-F) in the presence (B, C, E and F) of antibacterial micelles from polymer 6 were shown in the Figure S13 in the Supporting Information. The antibacterial mechanism is further demonstrated in Scheme 2.


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Scheme 2. Illustration of Antibacterial Mechanism of Polymer 6 Micelles. a)

a)

(A) Electrostatic interaction between negatively charged bacteria and positively charged

antibacterial micelles. (B) The antibacterial micelles adhere to the bacteria. (C) The membrane of bacteria is pierced by the micelles. (D) Rupture of bacteria membrane. The left-up picture in (A) high-lights the interaction between the membrane of bacteria and the antibacterial micelle. In vitro Enzymatic Biodegradation. PLLA is a kind of enzymatically degradable materials.42-44 The PLLA block of the micelle can be degraded into short segments even the Llactic acid once mixed with the lipase. Once the PLLA core is degraded, the polypeptide coronas can stretch and the micelles can be dissociated. The degradation degree is related to the


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concentration of the micelles in the solution, so the degradation process can be evaluated by measuring the decrease in the derived count rate with time. According to the degradation graph shown in Figure 4, the PLLA block was hydrolyzed quickly because the derived count rate decreased sharply. 100 (B)

(a) without lipase

-1

(b) with 0.025 µg mL lipase -1

(c) with 0.050 µg mL lipase -1

(d) with 0.100 µg mL lipase

Intensity (%)

100 (A) 90 80 70 60 50 40 30 20 10 0

Intensity (%)

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-1

(b) with 0.025 µg mL lipase

90 80 70 60

-1

(c) with 0.050 µg mL lipase

50 40

10

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30

Time (h)

40

50

-1

(d) with 0.100 µg mL lipase

0.0

0.5

1.0

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Figure 4. (A) Count rate variation of polymer 6 micelles during enzyme-catalyzed degradation. The function is determined by DLS at 37 °C and pH 7.4. The concentration of micelles is 30 µg mL-1. (B) Zoom-in of (A) between 0 and 2.5 h. The blank curve in Figure 4(A, a) indicated that the structure of the polypeptide-based micelles was stable and the self-degradation did not happen in the absence of lipase. In contrast, the micelles were degraded quickly in the presence of lipase (curves b, c and d in Figure 4A). In the presence of 50 µg mL-1 of lipase, more than 40% of micelles were degraded within 3 h. After 48 h, about 60% of micelles were degraded. When the concentration of lipase is 100 µg mL-1, the degradation rate is much faster than that at low lower lipase concentrations. CMC study of antibacterial micelles. CMC is defined as the lowest concentration of polymers to form micelles in water. To answer the question of whether the individual polymer


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chains or the polypeptide-based micelles kill bacteria in aqueous solution, we designed an experiment by comparing the CMC with the MIC value of polymer 6. As shown in Figure 5, the CMC is 13.2 µg mL-1, which is lower than the MIC50 value (40 µg mL-1), indicating that it is the polymer micelle that has antibacterial activity. 60000 55000 50000

Intensity

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45000 40000 35000 30000 25000 20000

CMC = 13.2 µg mL -0.4

0.0

0.4

0.8

1.2

1.6

2.0

lg[c/(µg mL-1)]

2.4

-1

2.8

Figure 5. Determination of the critical micellization concentration of polymer 6. Cytotoxicity Study. We also evaluated the cytotoxicity of polypeptide-based micelles by using CCK-8 assay against normal liver cells (L02) over 24 h. The concentration of micelles varies from 25 μg mL-1 to 100 μg mL-1, respectively. Below 50 μg mL-1, the antibacterial polypeptide-based micelles didn’t show obvious cytotoxicity (Figure 6). This is because the micellar morphology may decrease the cytotoxicity, and improve the blood compatibility to eukaryotic cells.30


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-1

25 µg mL -1 50 µg mL -1 100 µ g mL

140

Relative Cell Viability (% control)

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120

37.5 µg mL -1 75 µg mL

-1

100 80 60 40 20 0

12

Time (h)

24

Figure 6. Cytotoxicity studies of polypeptide-based micelles against liver L02 cells at different polymer concentrations. The relative cell viabilities were determined by CCK-8 assays (n = 5). CONCLUSIONS In summary, we have designed and synthesized a new PLLA and peptide-based copolymer [PLLA31-b-poly(Phe24-stat-Lys36)], which can self-assemble into micelles possessing strong positive charges (+ 51.8 mV) and excellent antibacterial activities against both Gram-negative and Gram-positive bacteria. The antibacterial mechanism, which shows the micelles have a physically damaging process by piercing the membrane of bacteria and causing bacteria contents flowing out and death, was directly observed by TEM. Moreover, the cationic groups in the antibacterial micelles can interact with the anionic groups in the cell wall, which possibly lead to a disturbance of the cations presented in the outer wall. This physico-chemical effect will enhance the antibacterial activity. The TEM evidence for the antibacterial mechanism of the polypeptide-based micelles may strengthen the confidence of scientists for designing nextgeneration antibacterial agents to fight against drug-resistant bacteria.


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ASSOCIATED CONTENT Supporting Information. Experimental details, Figures S1-S13 for additional 1H NMR spectra and calculations, DLS study, TEM and SEM images, and determination of MIC90, MIC99 MIC99.99. This information is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION * Corresponding author Email: [email protected] Author Contributions §

YX and TS contributed equally to this work.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT JD is supported by NSFC (21174107, 21374080, 21674081 and 21611130175), Shanghai 1000 Talents Plan, 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 (BNLMS20140127). Mingzhi Wang and Jingyi Gao are thanked for the help in the experiments. The reviewers of this manuscript are acknowledged for their valuable comments and suggestions. REFERENCES (1) Coates, A.; Hu, Y.; Bax, R.; Page, C. Nat. Rev. Drug Discovery 2002, 1, 895-910.


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