Highly Effective Antibacterial Vesicles Based on Peptide-Mimetic

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Highly Effective Antibacterial Vesicles Based on PeptideMimetic Alternating Copolymers for Bone Repair Chuncai Zhou, Yue Yuan, Panyu Zhou, Fangyingkai Wang, Yuanxiu Hong, Nuosha Wang, Shuogui Xu, and Jianzhong Du Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b01209 • Publication Date (Web): 11 Oct 2017 Downloaded from http://pubs.acs.org on October 13, 2017

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Highly Effective Antibacterial Vesicles Based on Peptide-Mimetic Alternating Copolymers for Bone Repair Chuncai Zhou†,‡,§, Yue Yuan‡,§, Panyu Zhou‖,§, Fangyingkai Wang‡, Yuanxiu Hong‡, Nuosha Wang‡, Shuogui Xu*,‖, 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, Tongji

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

Changhai Hospital, Department of Emergency, the Second Military Medical University, 168

Changhai Road, Shanghai 200433, China KEYWORDS: antimicrobial peptides; alternating copolymers; self-assembly; vesicles; bone repair

ACS Paragon Plus Environment

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ABSTRACT. It is an important challenge for bone repair to effectively deliver growth factors and at the same time to prevent and cure inflammation without obvious pathogen resistance. Herein, we designed a kind of antibacterial peptide-mimetic alternating copolymers (PMACs) to effectively inhibit and kill both Gram-positive and Gram-negative bacteria. The minimum inhibition concentrations (MICs) of the PMACs against E. coli and S. aureus are 8.0 µg/mL, which are much lower than that of antibacterial peptides synthesized by other methods such as widely used ring-opening polymerization of N-carboxyanhydride (NCA). Furthermore, the PMACs can self-assemble into polymer vesicles (polymersomes) in pure water with low cytotoxicity (IC50 > 1000 µg/mL), which can encapsulate growth factors in aqueous solution and release them during long-term antibacterial process for facilitating bone repair. We also find that the alternating structure is essential for the excellent antibacterial activity. The in vivo tests in rabbits confirmed that the growth-factor-encapsulated antibacterial vesicles have better bone repair ability compared with control groups without antibacterial vesicles. Overall, we have provided a novel method for designing PMAC-based highly effective intrinsically antibacterial vesicles which may have promising biomedical applications in the future.

Introduction. Bacterial infection from surgery is a serious problem because a complete cure requires a long period of repeated antibiotic therapy with a high cost and a risk of chronic infection.1,2 Several approaches have been used to solve this problem.3,4 Among them, traditional antibiotics are widely used to cure bacterial infections.5,6 However, the abuse of traditional antibiotics has led to the generation of drug-resistant pathogens, such as Methicillin-resistant Staphylococci aureus (MRSA), Vancomycin-resistant Enterococcus (VRE) and so on.

7,8


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Therefore, it is urgent to develop alternative antibacterial agents with excellent antibacterial activity but less pathogen resistance. Natural antibacterial peptide is an ideal host defense material because it can inhibit and kill bacteria, virus, and fungi without obvious pathogen resistance.9-11 Most of antibacterial peptides are cationic, and have hydrophilic amino acid residues (such as lysine or argine) and hydrophobic acid residues (such as phenylalanine, leucine, etc.).12-14 However, the production of antibacterial peptides by isolation and purification from natural sources such as epithelial or plant cells is an extremely tedious task, giving low yield and high cost to scale up.3,15 Therefore, various antibacterial peptides and peptide mimetics were chemically synthesized.16,17 For example, Tew and coworkers synthesized mimics of antimicrobial peptides with randomly copolymerized amino acid side chains.18 Gellman’s group synthesized antibacterial Nylon-3 copolymers as host-defense peptide mimics.19 Joy’s group reported bactericidal peptidomimetic polyurethanes with excellent selectivity against E. coli, and coumarin polyesters with pendant cationic amine groups which showed excellent antimicrobial activity against P. aeruginosa.20,21 However, it is still an important challenge to achieve highly effective antibacterial performance and low toxicity of antibacterial peptides. The bone repair is a slow and complicated physiological process involving the interaction of cells and cytokines.22 Growth factors are very important to the whole process as they can promote the cellular proliferation, the differentiation, and the synthesis of extracellular matrix during the fracture repair.23 Thus, the external growth factors are widely used to regulate bone repairing and the transportation and release of these regulators can be achieved through different ways.24-27 However, the growth factors may be degraded in vivo without the protection of


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carriers. Therefore, it would be ideal to transport growth factors using an inherently antibacterial delivery vehicle. Herein, we propose a new method for designing and synthesizing highly effective antibacterial materials with a mimetic peptide structure: peptide-mimetic alternating copolymers (PMACs). The PMACs were synthesized by the copolymerization of ε-Z-Lysine with hexamethylene diisocyanate (HDI), as shown in Figure 1. The copolymers have the similar structure to the cationic antimicrobial peptides, i.e., hydrophilic amino acid residues (lysine residues) and a hydrophobic part (hexamethylene group). These PMACs have excellent antibacterial ability against bacteria. Furthermore, the PMACs have lower toxicity to human cells and high selectivity compared with a range of current synthetic antibacterial peptides.20,28 Moreover, the PMACs can self-assemble into polymer vesicles with inherent antibacterial activity for transporting growth factors during bone repairing process. Therefore, two important missions in the bone repairing can be finished by one PMAC vesicle (Figure 2).


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Figure 1. Synthetic route towards Peptide-Mimetic Alternating Copolymers (PMACs) with excellent antibacterial activity.


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Figure 2. Engineering dual missions in one peptide-mimetic alternating copolymer (PMAC) vesicle for bone repairing: antibacterial and delivery of growth factor. The positively charged PMAC vesicles can attack negatively charged bacteria, impale and penetrate bacteria membrane, and finally kill bacteria. In the meanwhile, the encapsulated growth factors can be released from vesicles for facilitating bone regeneration. The growth factors can be also released in the healthy bone defects due to the degradation of vesicles.

EXPERIMENTAL SECTION Materials. N-ε-Benzyloxycarbonyl-L-lysine, hydrogen bromide (30% in acetic acid) were purchased from Shanghai Hanhong Chemical Co. Ltd. Hexamethylene diisocyanate (HDI), N,Ndimethylformamide (DMF), dimethylsulfoxide (DMSO) and 33% HBr/CH3COOH were


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purchased from Aladdin Industrial Corporation. NaOH, HCl (37%), diethyl ether, acetone and dialysis tubing with molecular weight cutoff from 8 000 to 14 000 were purchased from SinoPharm Group Co. Ltd (SCRC, Shanghai, China). The chemicals are not further purified unless otherwise specified. Gram-positive bacterium S. aureus (ATCC29213) and Gramnegative bacterium E. coli (ATCC35218) were purchased from Nanjing Bianzhen biological technology Co., Ltd. Synthesis of Monomer. The ε-Z-Lysine (50.00 g, 178.57 mmol) was added to DI water (500 mL) in a 1000 mL of beaker and stirred at room temperature. NaOH (7.140 g, 178.55 mmol) were dissolved in 50 mL of DI water and dropped into the mixture in 30 mins and then reacted for 2 h at room temperature. After filtration the solution was dried by rotary evaporation to afford a white solid. Yield: 99.0%. Then the ε-Z-Lysine (50.00 g) was dissolved in 300 mL of DMSO and stirred for 1 h. The HDI (13.90 g) was added in the solution and reacted at room temperature for 4 h. The mixture was poured to 500 mL of DI water. Then HCl (1.0 M) was dropped into the mixture to adjust the solution pH to 3.5. The solution was filtered and washed by DI water three times. The solid product (M) was dried by rotary evaporation. 1H NMR was shown in Figure S1 in the Supporting Information. Yield: 90%. Synthesis of PMACs. The monomer M (5.000 g) was added into 200 mL of DMF and stirred for 2 h. Then HDI (0.577 g) was charged into the mixture, reacted for 4 h at room temperature and 60 oC to continue the reaction for 4 h. DMF was removed by rotary evaporation. The crude product was washed by DI water for several times. The white powder was obtained by freeze-


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drying overnight to afford PMAC 1. The corresponding 1H NMR spectrum was shown in Figure S2 in the Supporting Information. Yield: 90%. PMACs 2-4 were synthesized according to the similar procedure by adding different amount of HDI (0.8650, 1.010 and 1.082 g, respectively). The corresponding 1H NMR spectra were shown in Figures S3-S5 in the Supporting Information, respectively. Antimicrobial Experiments. The antibacterial tests were performed with E. coli (ATCC35218) and S. aureus (ATCC29213) grown in the LB medium. The final result of each experiment was obtained from independent assays performed in triplicates.29-31 MIC100 values were determined by microdilution assay performed in sterilized 96-well plates in a final volume of 200 µL as follows: A 100 µL of LB medium containing bacteria were added to 100 µL of culture medium containing the peptide-mimetic polymers. Inhibition of proliferation was determined by optical density measurements (620 nm) after incubation overnight at 37 °C. The LB medium (100 µL) containing bacteria without copolymers were used as controls and the experiments were repeated at least three times. In addition, the MIC100 values were obtained based on the dynamic antibacterial results using a colony formation assay.32 Briefly, PMAC 4 vesicles solution (30 mg mL−1) was diluted to different concentrations with LB. Then 1.0 mL of these solutions and 20 µL of microorganism suspension were mixed in a conical flask at 37 °C. The optical densities of the mixtures were tested at various time intervals. The control is broth-containing bacteria cells without PMAC 4. Cellular Viability Test by CCK-8 Assay. The cellular viability was determined by the Cell Counting Kit-8 assay (CCK-8, Dojindo, Japan). L02 cells (human liver normal cell line) were seeded with equal density in each well of 96-well plates (4000 cells per well) in 100 µL of


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Dulbecco’s Modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) for 24 h at 37 °C in a humidified 5% CO2-containing atmosphere. Then 20 µL of solutions containing 62, 125, 250, 500 and 40 µg/mL of PMAC 4 vesicles were added and incubated with cells for another 24 h, respectively. Untreated cells were used as a control group. At the end of the treatment, CCK-8 dye was added to each well and the plates were incubated for an additional 1 h at 37 °C. Subsequently, the absorbance was measured by dual wavelength spectrophotometry at 450 nm and 630 nm using a microplate reader. Each treatment was repeated 5 times. The relative cell viability (%) was determined by comparing the absorbance at 450 nm with control wells containing only the normal L02 cells and the cell culture medium. Degradation of PMAC 4 vesicles. This experiment was conducted to evaluate the degradation degree of the peptide-mimetic polymer chains in the presence of lipase. The biodegradation of PMAC 4 was monitored by Dynamic Light Scattering (DLS). The aqueous lipase solution (0.91 mg/mL) was mixed with the vesicle solution and then put the mixture into an incubator at 37 °C. The count rate which corresponds to the degradation degree was detected at regular intervals by DLS. Vesicles in 0.1 M PBS solution were measured as controls in the same way. Surgical Procedures. This study followed the NIH guidelines for the care and use of laboratory animals (NIH Publication No. 85-23 Rev. 1985) and was approved by the Research Center for Laboratory Animal of The Second Military Medical University of China. The rabbit hindlimb muscle pocket model on the right leg was used to investigate the activity of ectopic bone formation of scaffolds combined with rhBMP-2 and antibacterial vesicles. The radical 20 mm defect models with rhBMP-2-loaded PMAC 4 vesicles were prepared in the following procedures. First, 0.500 mg of PMAC copolymer and 5.000 mg of rhBMP-2 were added in a beaker and then 10.0 mL of deionized water was added dropwise into the mixture and stirred


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continuously over 30 min. The rhBMP-2-loaded PMAC vesicles were dialyzed against tris buffer (0.01 M, pH 7.4) for 24 h to remove the unloaded growth factors. After that, the radical defect models were immersed into the prepared rhBMP-2-loaded vesicles solution for 4 hours and then freeze-dried quickly. The prepared radical models should be stored at -20 ℃. The radical defect models with rhBMP-2 only were prepared in the similar way. Scaffolds were divided into two groups: 1) with rhBMP-2; 2) with rhBMP-2-loaded PMAC 4 vesicles (50 µg/mL). Determination of Critical Vesiculation Concentration (CVC). The CVC is defined as the lowest concentration of polymers to form vesicles in water. We used pyrene as the probe to detect the vesicle formation. Pyrene (3.0 mg, 15 µmol) was dissolved in acetone (25 mL) as the initial solution of pyrene. Eleven centrifuge tubes were prepared to be filled with 10 µL of pyrene solution each. In order to get rid of the acetone, the eleven centrifuge tubes were put in the fuming cupboard overnight. The PMAC vesicles solution was diluted with deionized water into 11 different concentrations and then 4.0 mL of each was added into the centrifuge tubes, stirring overnight. Fluorescence of each solution was recorded by exciting samples at 334 nm, using a 5 nm slit width for excitation and a 5 nm slit width for emission. The samples were scanned by emission wavelengths from 350 to 500 nm. The intensities of the I1 (372.2 nm) vibronic bands were used to evaluate each sample. The intensity values were plotted against the log of the concentration of each vesicle. The CVC was obtained from the intersection of two regression lines programmed from the linear portions of the graphs. The CVC results were shown in Figure S10 in the Supporting Information.


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Statistical Analysis. The statistical analysis was conducted for all data. The data were presented as the mean ± standard deviation. Statistical analysis was conducted by SPASS 13.0 software. It is considered as a statistically significant difference when p < 0.05. Characterization. 1

H NMR. 1H NMR spectra were recorded using a Bruker AV 400 MHz spectrometer at room

temperature with DMSO-d6 and D2O as the solvent. Please note that the copolymers are difficult to be dissolved in organic solvent, such as THF, CHCl3 and DMF. DLS. DLS studies of aqueous polymer vesicle solutions were performed using a Nano-ZS 90 Nanosizer (Malvern Instruments Ltd., Worcestershire, UK) at a fixed scattering angle of 90°. The data were processed by cumulative analysis of the experimental correlation function, and particle diameters were calculated from the computed diffusion coefficients using the Stokes– Einstein equation. Each reported measurement was conducted for three runs. Zeta Potential Studies. The ζ-potential studies were conducted on a ZETA SIZER Nano series instrument (Malvern Instruments) at 25 oC. The software is Dispersion Technology Software (version 5.03). Transmission Electron Microscopy Studies. TEM was used to observe the formation of PMAC vesicles and the morphological changes of E. coli and S. aureus with PMAC. TEM images of vesicles and microorganisms before and after treatment by the vesicles were taken with a JEOL JEM-2100F instrument equipped with a Gatan 894 Ultrascan 1 k CCD camera at 200 kV.33 To prepare the sample of PMAC vesicles without bacteria, 5 µL of diluted vesicles suspension was dropped onto a carbon-coated copper grid and stained with phosphotungstic acid (1%, pH 5-


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6). The water droplet was allowed to evaporate slowly under ambient conditions before measurement. To prepare TEM samples of microorganism treated by vesicles, the microorganism (1.5 mL, 10 µM) in PBS solution was incubated with 0.5 mL of PMAC 4 vesicles solution at lethal doses (20 µg/mL) at 37 oC for 2 hours. The mixture was then centrifuged for 10 minutes at 5000 rpm to remove the supernatant. Then the mixture was added into PBS (pH 7.0, 0.5 mL) with 2.5% glutaraldehyde, and incubated overnight at 4 °C for fixation. Finally, the mixture was washed using the phosphate buffer for three times. The prepared samples (3 µL) were dropped onto a carbon-coated copper grid and dried at ambient environment. The samples were stained by phosphotungstic acid (1%, pH 5-6) solution for 1 min. Then a filter paper was used to blot the excess PTA solution. The samples were dried under ambient environment overnight. Atomic Force Microscopy (AFM) Studies. AFM was employed to verify the hollow structure (height contrast) of the polymer vesicles. Polymer vesicles solution was diluted to 30 µg mL-1 and dropped onto the fresh silicon wafer which was firstly washed in the acetone under ultrasound for 10 min. After being dried at room temperature, the observation could be conducted on a Seiko (SPA-300 HV) instrument operating in tapping mode at 200−400 kHz drive frequency. Three-Dimensional Micro-CT Imaging. Further assay was performed using a micro-computed tomographic imaging system (GE Explore Locus SP microCT, US). The defective radius and adjoining ulna were scanned; 3D µCT images were reconstructed then using Microview 2.2 software to evaluate the repair process (GE Health Systems, U. S.). The defect sites were


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analyzed to calculate the bone volume (BV) and bone mineral density (BMD) (n = 5 rabbits/batch).

RESULTS AND DISCUSSION Synthesis and Characterization of Peptide-Mimetic Alternating Copolymers. The synthetic route to PMACs is shown in Figure 1. First, ε-Z-Lysine was neutralized by NaOH to form sodium ε-Z-Lysine. The formation of –COONa significantly increases the solubility of ε-ZLysine in DMSO and protects –COOH group. Then the –NH2 group of sodium ε-Z-Lysine reacts with HDI to afford dicarboxyl monomer M. Second, M reacts with HDI at room temperature to form protected PMACs. After deprotection in HBr/CH3COOH, the final cationic PMACs are obtained. Simply tuning the ratio of M to HDI, PMACs with various degree of polymerization were obtained (n = 4, 6, 12 and 30, see Figure 1). The 1H NMR analysis confirmed successful synthesis of PMACs (Figures S1-S9 and Tables S1-S5 in the Supporting Information). These PMACs have similar structure to natural cationic antimicrobial peptides, i.e., hydrophilic amino acid residues (lysine residues) and a hydrophobic group (hexamethylene groups). The molecular weight and molecular weight distribution are not obtained because the copolymers don’t dissolve in organic solvents such as THF, CHCl3 and DMF. Determination of the critical vesicle formation concentration of vesicles. Like block copolymers,34,35 homopolymers36 and statistical copolymers,37 PMACs can also self-assemble into vesicles in aqueous solution due to their inherent amphiphilicity. It is noteworthy that PMACs can be directly dissolved in pure water to form polymer vesicles without the aid of organic solvents, which will greatly facilitate the facile encapsulation of growth factors in pure


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water. The critical vesiculation concentration (CVC) for PMAC 4 in water is 14.6 µg mL-1 (Figure S10 in the Supporting Information). Analysis of the Diameter of the Antibacterial Micelles by TEM and DLS. TEM study confirmed the hollow structure of vesicles self-assembled from PMAC 4 (the degree of polymerization is 30) with a mean diameter of 429 ± 115 nm (Figure 3). The membrane thickness is 18.2 nm as calculated according to our previously reported folded membrane model of nanosheets.38 The TEM image is a function of electron density transmitting the membrane of the vesicle. The vertical thickness (L) can be treated as a function of the membrane thickness (d) in the case of folded membrane of the vesicles. The folded membrane is simplified to such a model, in which the lower part was the original semiannulus and the upper part consists of one semicircle and four quarter annuli (Figure 3C). The radius of the semicircle is equal to d. The outer and inner radii and the thickness of the annulus are R1, R2 and d, respectively. A piecewise equation is presented in the Supporting Information for detailed calculation of membrane thickness. DLS study revealed that the hydrodynamic diameter (Dh) of polymer vesicles prepared by PMAC 4 was 279 nm with a PDI of 0.231, as shown in Figure S10 and Figure S11 in the Supporting Information. Also, DLS study showed that vesicles prepared from PMACs 4 have better size distribution than others. AFM was used to further investigate the morphology of the vesicles (Figure S12 in the Supporting Information). The diameter and the height of the vesicles are 360 nm and 53 nm, respectively. This is because the soft and deformable vesicle collapsed on the silicon substrate to show a large diameter/height ratio of 6.87, indicating a hollow structure. Zeta potential study of the vesicles in Table S6 in the Supporting Information shows that all the vesicles are positively charged (+ 25.1 mV for PMAC 4 vesicles).


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Figure 3. TEM images (A-B) and membrane thickness analysis (C-E) of vesicles from PMAC 4.

Antibacterial Activity. The minimal inhibitory concentration (MIC) is generally defined as the minimum concentration of an antimicrobial agent at which no visible growth of microbes is observed, which is commonly used to evaluate the activity of antibacterial agents. We evaluated the MICs of the PMACs against both Gram-positive bacteria and Gram-negative bacteria by two different methods. First, the critical dilution method using polystyrene micro-assay plates (96-well Microtest).39 Briefly, microplates loaded with two-fold serial dilutions of PMACs in Luria-Bertani broth (LB


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broth) were seeded with approximately 1×105 CFU per well. Microplates were incubated at 37 o

C for 18 h. MICs were expressed in milligram per milliliter and correspond to the lowest

concentration that limited the development of the turbidity after 18 h. The MICs results showed that all the four kinds of PMACs have high activity against both Gram-negative and grampositive bacteria. Among them, PMAC 4 has the lowest MIC100 values against E. coli and S. aureus (8.0 µg/mL, see Table 1), which is lower than many natural antibacterial peptides.40 It is noteworthy that the MICs are lower than the critical vesiculation concentration (CVC) of PMACs. For example, the CVC of PMAC 4 in water is 14.6 µg mL-1 (see Figure S10 in the Supporting Information). This means that once the PMACs form vesicles in water, they will be able to effectively inhibit and kill bacteria. Table 1. MIC values of different PMACs against E. coli and S. aureus by 96-well Microtest Bacterial Species

Concentration (µg/mL)

1 1

E. coli S. aureus

125 62.5 31.25 15.6 7.8 3.9 + + + + -

2

E. coli

+

+

-

-

-

-

2

S. aureus

+

+

-

-

-

-

3

E. coli

+

+

+

+

-

-

3

S. aureus

+

+

+

+

-

-

4

E. coli

+

+

+

+

+

-

4

S. aureus

+

+

+

+

+

-

PMAC

“+” represents no more bacteria survive at particular concentration, “-” represents bacteria still exists.

Usually, antibacterial peptides are composed of hydrophilic part (positive charge to adsorb with bacterial cells) and hydrophobic part (to insert into the membrane of bacterial cells). The hydrophobic non-amino-acid organic molecule in PMACs replaced the role of hydrophobic


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amino acid residue (such as phenylalanine) in natural antibacterial peptide, showing better antibacterial activity than antibacterial peptides. To evaluate the dynamic antibacterial activity of the mimetic peptide (PMAC 4), the MICs were measured using a colony formation assay.32 Antibacterial tests revealed the growth rates of bacteria under different conditions and the optical density indicated the viability of bacteria (Figure 4). The MICs of PMAC 4 against both Gram-negative E. coli and Gram-positive S. aureus were 8.0 µg/mL, showing excellent antibacterial activity. These results are consistent with the MIC values determined by 96-well Microtest (Table 1).

Figure 4. Dose-dependent growth inhibition of typical Gram-negative (E. coli) and Grampositive (S. aureus) bacteria in the presence of PMAC 4. OD: optical density. Control: no PMAC. Cytotoxicity Study. The PMACs (and the corresponding vesicles when the concentration is above CVC) have excellent antibacterial activity by destroying the membrane of bacterial cell. Thus, the cytotoxicity of PMAC 4 vesicles was evaluated by a Cell Counting Kit-8 (CCK-8) assay against normal liver cells (L02). IC50 is defined as the concentration of copolymer solution where the cell viability decreases to the half maximal. It is noteworthy that the L02 cells remained high metabolic activities with an IC50 value > 1000 µg/mL after 24 h (Figure S13 in


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the Supporting Information). Therefore, the PMACs and their vesicles can be used in a broad range of concentrations for antibacterial applications. Degradation of PMAC 4 vesicles. It is important for polymer vesicles to be degraded in the presence of enzyme once the vesicles finished the delivering duties in vivo. The biodegradation of PMAC vesicles was tracked by DLS through measuring the decrease in the scattering intensity with time (Figure S14).41 The derived count rate of PMAC 4 vesicles was decreased from 1619.2 (100%) to 963.1 (59.5%), indicating that the vesicles were dissociated and degraded in the presence of lipase. Antibacterial Mechanism of Antibacterial Vesicles by TEM. To explore the mechanism of the antibacterial action of the PMAC vesicles, we investigated the morphological changes of E. coli and S. aureus before and after incubation with PMAC 4 vesicles for 8 h at the lethal dose (20 µg/mL) which is above both MIC and CVC values. TEM analysis in Figure 5B and D revealed that the cell membranes of the bacteria were damaged, and cell lysis was observed after treatment with the vesicles. As illustrated in Figure 5E and F, We hypothesize that the positively charged PMACs in the vesicles can interact with the negatively charged bacterial cell wall via electrostatic interactions.42 Then the PMACs may insert into the cell membrane (especially for Gram-negative bacteria, B) leading to partial merging of the lipid with the PMAC vesicles. Finally, the pores form on the cell membrane of bacteria and the leakage of the cell contents results in the death of bacteria.43,44 The PMACs have better antibacterial activity against both Gram-negative E. coli and Grampositive S. aureus than peptides prepared by NCA method and lots of natural antibacterial peptides.32 Possibly, the positive charge (lysine) and hydrophobic amino acid residues (which are one of predominant component in antibacterial process) of antibacterial peptide prepared by


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NCA method are not evenly distributed in the peptide chains due to the difference in the reaction activity of the NCA monomers. By contrast, the PMACs have well-defined alternating lysine/hexamethylene structure, which makes the hydrophobic groups be limited on the surface of the bacteria cell (Figure 5F). This alternating structure may increase the efficiency of the hydrophobic group inserting into the membrane of bacteria cell because a higher MIC value of 32 µg/mL was obtained for the statistical mimetic polypeptide copolymer in a control experiment (see Figure S15 in the Supporting Information for comparison). We hypothesize that this is the main reason for the excellent antibacterial activity of PMACs.

Figure 5. Comparative TEM images of microbe in the absence and presence of antibacterial PMAC 4 vesicles and the proposed antibacterial mechanism. (A and B) E. coli before and after the incubation with vesicles; (C and D) S. aureus before and after the incubation with the


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vesicles. (E and F) Proposed antibacterial mechanism of PMAC vesicles (above CVC) and PMAC (below CVC). Bone Repair Tests. The PMAC vesicles can encapsulate and deliver necessary growth factors for facilitating bone repair and long-term antibacterial application. After initial optimization on the scaffold formulation using in vitro cytocompatibility testing and in vivo ectopic bone formation evaluation, the ability of the bone xenograft scaffolds with rhBMP-2-loaded PMAC 4 vesicles to repair critical-sized bone defects were examined. A radical 20 mm defect model was used in rabbits. Micro-CT, bone mineral content and bone mineral density were used to evaluate the repair of bone defects with scaffolds at 4 and 6 weeks after implantation. Representative micro-CT images of 3D structures of repaired radii showed that there were new bones formed on the defects. The results of 6 weeks were better than that at 4 weeks (Figure 6AF). Quantitative analysis of radiopacity in 3D micro-CT images was further employed. The regenerated bone volumes (BV) and bone mineral density (BMD) within the defect were also calculated to precisely evaluate the repair of bone defects. The “rhBMP-2 + Vesicles” group exhibited bigger bone volume than the rhBMP-2 group at both 4 and 6 weeks (Figure 6H). A significant increase in bone mineral density value was observed with PMAC 4 vesicles than the rhBMP-2 group (no vesicles, see Figure 6G). The results indicated that the PMAC 4 vesicles may not only replace the traditional antibiotics in the process of bone repair, but also boost the regeneration of new bone.


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Figure 6. Rabbits critical-sized bone defects repair process using vesicle/rhBMP-2 loaded scaffolds. (A to F) 3D Micro-CT reconstructed images of rabbit segmental radius at 4 and 6 weeks with different implants: (A) BMP, 4 w; (B) BMP + Vesicles, 4 w; (D) BMP, 6 w; (E) BMP + Vesicles, 6 w; (C and F) controls without any implants; G) Quantitative analysis of mineralized new bone formation from SRuCT images: bone mineral density at 4 and 6 weeks; (F) Regenerated bone volume at 4 and 6 weeks. n = 5, p < 0.05. CONCLUSIONS In summary, we have developed a novel antibacterial peptide-mimetic alternating copolymer which can directly self-assemble into antibacterial vesicles in pure water. Both the copolymer


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and the vesicles exhibit low cytotoxicity and excellent antibacterial efficacy against Grampositive and Gram-negative bacteria. Furthermore, the antibacterial vesicles are able to encapsulate growth factors to facilitate the bone repairing process by long-term release growth factors and inherent antibacterial. The in vivo experiments confirm that the PMAC vesicles can not only take place of traditional antibiotics in the bone tissue regenerations, but also speed up the regeneration of new bone. Overall, these low cytotoxic, biocompatible, highly active antibacterial PMAC vesicles may have potential clinical applications in bone repair process. ASSOCIATED CONTENT Supporting Information.

Full synthetic and characterization details, Figures S1-S15 and

Tables S1-S6. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION * Corresponding author Email: [email protected] ORCID Chuncai Zhou: 0000-0002-8176-4429 Yue Yuan: 0000-0002-5438-4842 Panyu Zhou: 0000-0001-7956-2994 Fangyingkai Wang: 0000-0001-7756-6561 Yuanxiu Hong: 0000-0002-8572-2488 Nuosha Wang: 0000-0001-8253-0360 Shuogui Xu: 0000-0001-5926-4391


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Jianzhong Du: 0000-0003-1889-5669

Author Contributions §These authors contributed equally. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work is supported by NSFC (21611130175, 21374080, 21674081, 21274110 and 21174107), Shanghai International Scientific Collaboration Fund (15230724500), Shanghai 1000 Talents Plan, and the Fundamental Research Funds for the Central Universities (1500219107). REFERENCES (1) Darouiche, R. O. Treatment of Infections Associated with Surgical Implants. N. Engl. J. Med. 2004, 350, 1422-1429. (2) Whitehouse, J. D. M. D.; Friedman, N. D. M.; Kirkland, K. B. M. D.; Richardson, W. J. M. D.; Sexton, D. J. M. D. The Impact of Surgical‐Site Infections Following Orthopedic Surgery at a Community Hospital and a University Hospital: Adverse Quality of Life, Excess Length of Stay, and Extra Cost. Infect. Control. Hosp. Epidemiol. 2002, 23, 183-189. (3) Choi, S.; Isaacs, A.; Clements, D.; Liu, D. H.; Kim, H.; Scott, R. W.; Winkler, J. D.; DeGrado, W. F. De novo Design and in vivo Activity of Conformationally Restrained Antimicrobial Arylamide Foldamers. Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 6968-6973.


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For Table of Contents Use Only

Highly Effective Antibacterial Vesicles Based on PeptideMimetic Alternating Copolymers for Bone Repair Chuncai Zhou, Yue Yuan, Panyu Zhou, Fangyingkai Wang, Yuanxiu Hong, Nuosha Wang, Shuogui Xu, and Jianzhong Du


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Highly Effective Antibacterial Vesicles Based on Peptide-Mimetic

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