Biodegradable, Biomimetic Elastomeric, Photoluminescent, and Broad

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Tissue Engineering and Regenerative Medicine

Biodegradable, Biomimetic Elastomeric, Photoluminescent and Broad-spectrum Antibacterial Polycitrate-Polypeptidebased Membrane towards Multifunctional Biomedical Implants Feng Li, Yajuan Su, Guofu Pi, Peter X Ma, and Bo Lei ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b00660 • Publication Date (Web): 25 Jun 2018 Downloaded from http://pubs.acs.org on June 26, 2018

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ACS Biomaterials Science & Engineering

Biodegradable, Biomimetic Elastomeric, Photoluminescent and Broad-spectrum Antibacterial Polycitrate-Polypeptide-based Membrane towards Multifunctional Biomedical Implants Feng Li a,1, Yajuan Su b, 1, Guofu Pi a,*, Peter X Ma d, Bo Lei b, c, e, f * a

Department of Orthopaedics, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052,

China b

Frontier Institute of Science and Technology, Xi’an Jiaotong University, Xi’an 710054, China

c

State Key Laboratory for Mechanical Behavior of Materials, Xi‫׳‬an Jiaotong University, Xi‫׳‬an 710054,

China d

Department of Biomedical Engineering, Macromolecular Science and Engineering Center, University of

Michigan, Department of Materials Science and Engineering, University of Michigan, Ann Arbor, MI 48109-1078, USA e

State Key Laboratory for Manufacturing Systems Engineering, Xi'an Jiaotong University, Xi'an 710054,

China f

Instrument Analysis Center , Xi’an Jiaotong University, Xi'an 710054, China

Abstract: Present biomedical membranes in guiding tissue regeneration applications are almost non-biodegradable or deficient in functionality. The development of biodegradable biomaterials with multifunctional properties including biomimetic elastomeric behavior, self-anti-infection, non-invasive monitoring and good biocompatibility, has attracted much attention. Here, we report a biodegradable and biocompatible polycitrate-(ε-polypeptide)-based (PCE) biomedical elastomers membrane with intrinsical broad-spectrum antibacterial activity and photoluminescent capacity for multifunctional guiding tissue regenerative applications. PCE showed highly elastomeric mechanical behavior (~300% elongation and ~100% recovery) and biomimetic mechanical properties against several native tissues. PCE film also

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possessed highly efficient broad-spectrum antibacterial activity in vitro and in vivo. The inherent photoluminescent properties of PCE film endowed their real-time non-invasive monitoring capacity in vivo. Owe to the biocompatible structure (polycitrate and natural polypeptide), PCE film demonstrated significantly high cytocompatibility and hemocompatibility in vitro and low inflammatory response in vivo. Our study may provide a new strategy to design next generation multifunctional biodegradable biomedical implants membrane for smart guiding tissue regenerative medicine applications. Keywords: multifunctional membrane, biomedical implant, biodegradable biomaterials, antibacterial, guiding tissue regeneration; Polymer-based biomedical implants play an important role in tissue replacement and tissue engineering, because of their easy fabrication and biocompatible feature.

1

The implanted biomaterials

include physical supported implants such as bone implants and artificial blood vessels, and functional devices such as pacemaker. 2 The physical supported implants are usually used to provide the functions of native tissues or repair local tissue defect. In recent years, the biomedical implants and devices showed important demands on the multifunctional biomaterials. However, current biomedical implants and devices only possess single function, therefore, development of multifunctional biomaterials has shown increased interests.3, 4 To meet the various requirements in biomedicine, several functions are needed including biomimetic elastomeric behavior, antimicrobial activity, real-time monitoring, linear degradation and biocompatibility.5 However, except the composites, most of biomedical polymer materials do not have these functions in the same structure. 6 Native tissues such as muscles, tendon and blood vessels usually showed high viscoelastic mechanical properties and high biocompatibility. 7 Therefore, recently biodegradable elastomeric polymers were developed and studied as biomedical implants used in regenerative medicine. 8 These polymers have


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biomimetic mechanical property and enhanced biocompatibility in various applications. For example, polycitrate and poly(glycerol)-based elastomers have been synthesized through a facile thermal polymerization method.

9, 10

To obtain the fluorescent polymer, amino acids were copolymerized with

poly(citrate) to form photoluminescent implants which showed the potential applications in bioimaging-guided tissue engineering.

11

To give the antibacterial properties for polymers, various

antibacterial drug or molecules were grafted on biomedical polymers.

12, 13

Although some interesting

results have been achieved in the design and application of biomedical elastomers, the inherent multifunctional elastomers with linear degradation profiles are few reported. Polycitrate-based (PC) biomedical elastomers have attracted much attention in regenerative medicine due to serval advantages including facile synthesis, linear biodegradation, biomimetic elastomeric behavior and good biocompatibility. 14,15 The remained chemical groups in the structure of PC such as carboxyls and hydroxyls make them easy to be functionalized through further chemical reaction. For example, through a copolymerization between siloxane and poly (citrate), poly (citrate-siloxane)-based elastomers was fabricated to improve their mechanical properties and osteogenic bioactivity.

16-18

On the other hand,

bacterial infection has been a major challenge in biomedical implants-related regenerative medicine.

19

However, current elastomeric polymer implants with high broad-spectrum antimicrobial activity are still unsatisfactory. In addition to the anti-infection ability, the invasive imaging of implants in vivo is also the important requirement to monitor their change.

20

Therefore, recently the photoluminescent PC-based

elastomeric polymers were developed for bioimaging application.

21

It is necessary and important to

develop elastomeric biomedical polymer implants with broad-spectrum antibacterial activity and photoluminescent activity. As compared with other antimicrobial biomaterials, antimicrobial peptides (AMPs) possess several



advantages such as high antibacterial, antiviral, antibiofilm and immunomodulatory activities. 22-25 As a low cost cationic polypeptide polymer produced by Streptomyces albulus, poly-ε-L-lysine (EPL) possesses a high broad-spectrum bacteria activity.26 In our previous study, we develop a multifunctional EPL-based poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P3/4HB) copolymers with high antibacterial activity and enhanced siRNA delivery ability. 27 In this study, we propose a copolymerization of EPL in the structure of PC polymer to fabricate a biodegradable multifunctional PC-EPL (PCE) bioelastomers with a broad-spectrum antibacterial activity and controlled photoluminescence. The physicochemical structure, biomimetic elastomeric behavior, linear degradation, antibacterial activity, in vivo bioimaging, biocompatibility of PCE elastomers were investigated in detail. RESULTS AND DISCUSSION Synthesis and characterization of PCE polymer. The synthesis and characterization of PCE prepolymer and elastomers are shown in Figure 1. The residual carboxyls in PC structure could be reacted with the amino group in EPL to form the amine bond, under the catalytic process of EDC and NHS (Fig.1A). After the chemical crosslinking process of –OH or NH2 under the presence of HDI, the elastomeric network was formed (Fig.1A). The chemical structure of PCE polymer was analyzed by 1H NMR and FTIR spectra (Figs.1B-C). The representative proton peaks attributed to PC and EPL could be found in 1H NMR (Fig.1B). The methylene peaks (-CH2- at 1.27, 1.54 and 3.97 ppm) and the peaks between 2.64-2.89 ppm (-CH2-) were assigned to 1.8-octanediol and citric acid respectively (Fig.1B). After reacted with EPL, the presented peaks at 1.9 and 3.5 ppm (-CH2) were belonged to EPL. Based on the 1H NMR of PCE, the actual molar ratio percent of EPL in PCE 1.0 was about 52.6%, The average molecular weight (Mw) of PC was about 5640 g/mol with a polydispersity (PDI) of 1.46. After the reaction of EPL, PCE 1.0 showed an average Mw of 8640 g/mol with a PDI of 1.51. The detailed structure parameters were


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shown in Table S1. In the FTIR spectra of PCE, the new peaks at 1640 cm-1 and 1560 cm-1 were assigned to the amine bonds in PCE, which indicated the formation of PCE polymer (Fig.1C). Biomimetic mechanical properties. Most of native tissues such as skin, muscle and blood vessels, show good elastomeric behavior and antifatigue property in vivo. 28 As advanced biomedical implants, the good shape-recovery ability is necessary for their successful application. Here, after copolymerization of EPL, the PCE elastomers still showed controlled elastomeric mechanical behavior (Figure 2). PCE 1.0 exhibited a quick elastomeric recovery performance after high stretching (Fig.2A). All PCE-based elastomers indicated a representative elastomeric stress-strain behavior (Fig.2B). No significant difference at tensile stress, modulus, elongation and recovery ratio for PCE 2.0, as compared to PCE 0 (Fig.2C). The tensile stress, modulus and elongation of samples were in the range of 5.1-6.5 MPa, 3.1-3.8 MPa, 255-305% respectively. It should be noted that the tensile stress of PCE elastomers was matched with native cartilage, pericardium, and coronary artery tissue.

29-31

Compared with PCE 0, the PCE 2.0 showed an increased

mechanical hysteresis loop after one cycle tension (Fig. S1), which suggested the decreased elastomeric recovery ability. The elastomeric strain of PCE was significant high compared with native tissues. These high elastomeric mechanical properties of PCE elastomers make them high promising for biomedical implants application. Biodegradation and bioimaging in vitro and in vivo. The controlled biodegradation of biomedical implants plays an important role in vivo long-term application.

32

The biodegradation for polyesters

polymer was usually related with their hydrophilicity. Therefore, we tested the hydrophilicity of PCE through analyzing the water contact angle. As shown in Fig.2D, pure PC film (PCE 0) showed a water contact angle about 88.5°which indicated its hydrophobic feature. As the copolymerization of EPL, the water contact angle of PCE was decreased significantly to 43.6°, suggesting their enhanced hydrophilicity.



The significantly increased hydrophilicity of PCE could be attributed to the fact that EPL was a highly hydrophilic polymer. The wide range of water contact angle may also affect the biodegradation of PCE elastomers. In this study, we measured the weight loss of PCE films through soaking them in PBS for 28 days. The results showed that the incorporation of EPL significantly improved the weight loss of PCE (Fig.2E). After 28 days soaking, PCE 0 and PCE 2.0 exhibited a weight loss of 27.8% and 37.6% respectively. The weight loss result of PCE was positively related with their hydrophilicity. To investigate the degradation behavior in soaking time, we made an exponential fitting on the weight loss curves (Fig.2F). It was very interesting that the weight loss of PCE film during the first 24 days was a typical exponential process, suggesting their controlled degradation in the long term. The biomedical implants with intrinsical photoluminescent ability could allow them real-time tracked in vivo, which has attracted much attention recently.

25

Our PCE elastomers show highly controlled

photoluminescent ability without adding any fluorescent dyes or quantum dots, as shown in Figure 3. Under the UV lamp at 365 nm, PCE0.5, PCE 1.0, PCE 2.0 showed a bright blue-green fluorescence, as compared to the poor fluorescence for PCE 0 (Fig.3A). No significant decrease of fluorescence was observed for PCE after six months storing at room temperature. The fluorescent intensity of PCE 0.5 film showed an increase after six months but was still in the same order of magnitude compared with the initial intensity. The maximum fluorescent emission wavelength of PCE was about 525 nm under the excitation at 365 nm (Fig.3B). There was no obvious decrease on fluorescent emission spectra of PCE after six months (Fig.3C). Compared with the commercial fluorescent dye (rhodamine), our PCE film demonstrated a significantly high fluorescent stability and no big decrease on fluorescent intensity was observed after 150 min excitation at 365 nm (Fig.3D). In addition, our PCE 1.0 elastomers showed an absolute quantum yield of 16%, further indicating their good photostability. The quantum yield of PCE 1.0 was also comparable to


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that of reported nonconjugated biodegradable polymers.

33, 34

The photoluminescent mechanism of PCE

was similar with that of poly(amido amine)-based polymers, although there was no any conjugated structure in these polymers.

33

Pure EPL does not show any photoluminescent ability under excitation

wavelength at 365 nm (Fig.S2). Figs.3E-F shows the in vivo bioimaging and real-time degradation performance of PCE films. As the increase of implantation time, the fluorescent signal was decreased gradually. The weight loss of implants was in accordance with the fluorescent decay, suggesting that the degradation of samples could be monitored by the real-time fluorescent imaging (Fig.3E). Broad-spectrum antimicrobial activity in vitro and in vivo. The microbial infection on biomedical implants has been one of major concerns in successful in vivo applications. 35 Therefore, in recent years, the antimicrobial biomaterials with broad-spectrum antibacterial activity have attracted much attention. In this study, we evaluated the antibacterial property of PCE elastomers using S. aureus, E. coli, P. aeruginosa, E. faecalis, as shown in Figure 4. Fig.4A shows the antibacterial efficiency of PCE at different concentrations of bacteria. Whatever the bacteria concentration (105-107 CFU/mL), PCE 0 exhibited limited antibacterial activity below 60%. As the incorporation of EPL, the antibacterial activity of PCE was significantly improved. At the low bacterial concentration of 105 CFU/mL, PCE 0.5 possessed high kill ratio above 96% and this value was decreased to 75%. PCE 1.0 and PCE 2.0 demonstrated significantly high antibacterial efficiency (above 96%) even at high bacterial concentration (107 CFU/mL). The in vitro bacterial growth images also indicated that PCE 1.0 and PCE 2.0 significantly inhibited the biofilm formation of S.aureus, as compared to PC and PCE 0 (Fig.4B). Compared to the in vitro analysis, the in vivo environment was more complicated due to various biomolecules and protein adsorption on implants. Therefore, it is very necessary to evaluate the in vivo antibacterial activity of biomedical implants. Here, we evaluate the antimicrobial activity of PCE using a



rodent subcutaneous infection model. After 5 days postimplantation with pre-seeded S.aureus, the samples were removed out to investigate the bacterial growth and activity. The bacteria from PCE 0 showed a quick growth after 24 hours culture, suggesting their low antibacterial activity (Fig.4C). Compared with PCE 0, no visible bacteria formation was observed on PCE 1.0, indicating the high antiinfection ability of PCE 1.0 (Fig.4C). The quantitative results also showed that PCE 0 and PCE 1.0 exhibited an average microbial load of 6.2 log CFU and 1.3 log CFU per sample respectively (Fig.4D). The further SEM images showed that the bacteria demonstrated a significant deformation on PCE 1.0 and no change was observed on PCE 0 (Fig.4E). These results further demonstrated that PCE 1.0 elastomers have excellent antibacterial activity in vitro and in vivo. Biocompatibility in vitro and in vivo. The conventional antibacterial biomaterials such as silver ion show potential cytotoxicity towards mammalian cells.

36

Our PCE showed high antibacterial activity but

low cytotoxicity on blood cell and myoblasts (Figure 5). The blood cell compatibility was analyzed by testing the hemolysis of samples. All PCE samples did not show significant hemolysis and the hemolysis percentage was only below 3% (Fig.5A), suggesting their high hemocompatibility with blood cells. The myoblast (C2C12) was also used to evaluate the cytocompatibility of PCE. The C2C12 cells were significantly increased after 5 days culture on PCE film, demonstrating their good cytocompatibility (Fig.5B). In addition, on day 1 to day 5, the cell viability on PCE 1.0 was significantly high, as compared to PCE 0 and PCE 2.0. The cell viability results may be attributed to the chemical structure of PCE polymers. The live-dead staining also demonstrated that no significant dead cells were observed on PCE 1.0 after 5 days culture (Fig.S4). It was well known that EPL was cationic peptide which was helpful for the cell attachment. However, too much EPL may have high positive density which would hurt the cell activity. The subcutaneous rat model was used to evaluate the in vivo biocompatibility of PCE elastomers. The H&E


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staining was employed to determine the tissue response of PCE after implantation for 4 weeks. As shown in Fig.5C, PCE 1.0 showed a significant low fibrous capsule formation compared with PCE 0, suggesting the low inflammatory response of PCE 1.0 (Fig.5C). The average fibrous capsule thickness around PCE 1.0 was about 49 µm (Fig.S3), which was significantly low, as compared to the PCE 0 film and the commercial poly (lactic-co-glycolic acid) (PLGA) implants (150 µm in thickness after 1 week).

37

The in vitro and in

vivo experiments clearly showed that PCE 1.0 possesses excellent biocompatibility and have a great potential for further regenerative medicine applications. The detailed in vivo performance on soft tissue regeneration should be further studied. CONCLUSION In summary, biodegradable and multifunctional elastomeric PCE film were synthesized through a facile thermal polymerization and further crosslinking process. PCE film exhibited highly elastomeric behaviors, biomimetic mechanical properties, and controlled biodegradation process. PCE film also demonstrated controlled photoluminescent ability and could be used for long-term in vivo degradation tracking imaging. The broad-spectrum antibacterial activity of PCE was also observed and was successful used for in vivo antiinfection. In addition, PCE 1.0 film presented good hemocompatibility, cellular biocompatibility and in vivo tissue compatibility. The biodegradable PCE 1.0 film with biomimetic mechanical property, intrinsical photoluminescence, broad-spectrum antibacterial activity and biocompatibility, may serve as an excellent multifunctional platform for smart regenerative medicine. EXPERIMENTAL SECTION Materials. Citric Acid (99%), 1, 8-Octanediol (98%), tetrahydrofuran (THF), dimethyl sulfoxide (DMSO), hydrochloric acid (37%), 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC, 99%), N-Hydroxysuccinimide (NHS, 98%), stannous octoate and hexamethyl diisocyanate (HDI) were



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obtained from Sigma-Aldrich. Poly-ε-L-lysine (EPL, Mn = 3500 Da) was received from Zhengzhou Bainafo Bioengineering Co., Ltd. (Henan, China). All chemicals were used as purchased without purification. Synthesis and characterizations of PCE polymers. The PC prepolymer was synthesized through a thermal polymerization of citric acid (CA), 1,8-octanediol (OD), according to our previous report.

38

The

PCE polymers were synthesized using PC and EPL through a EDC and NHS chemistry. Briefly, PC was dissolved in DMSO/H2O (4/1 volume) containing EDC and NHS, followed by adding EPL with various contents (COOH/NH2 mole ratio: 0, 0.5, 1.0, 2.0). The resulted solution was stirring for 48 h at room temperature, and purified via dialysis (MWCO 6000) for 2 days. The final samples were obtained after further freeze-drying for 2 days and the sample was denoted as PCE 0, PCE 0.5, PCE 1.0, PCE 2.0 respectively. The crosslinking elastomeric films were fabricated through a HDI cross-linking method. PCE polymer solution in DMSO (1 mg/ml) was reacted with HDI at a molar ratio of 0.5 (residual hydroxyls of pre-polymer), using stannous octoate as a catalyst (0.1wt %). The crosslinking reaction was performed at 50℃ for 2 h, followed by casting into a Teflon mold for solvents evaporating. The elastomeric PCE films were obtained through a post-polycondensation at 60℃ for 24 h. The structure of PCE was measured by the 1H nuclear magnetic resonance (1H NMR) instrument (Ascend 400 MHz， Bruker) using tetra-methyl-silane (TMS, 0.00 ppm) as the internal reference and Fourier transformation infrared (FT-IR) spectroscopy (NICOLET 6700, Thermo). The FT-IR spectra was performed through a KBr disk method and the spectra were collected at a scan resolution of 4 cm-1. The average molecular weight of PC and PCE polymer was obtained by a gel permeation chromatography (GPC, Waters, USA) using THF as the eluent, calibrated against linear polystyrene standards. Mechanical properties evaluations. The tensile mechanical tests were performed by a uniaxial tensile


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test (MTS CriterionTM Model 43, MTS) and the results were analyzed by the TestWorks4 software (MTS Systems corp., MN). Before test, the PCE film was cut to a size of 50 × 5.0 mm at length× width and the tensile stress-strain curves were obtained at rate of 50 mm/min. The tensile strength and elongation at break was calculated according to the stress-strain curves and the size of samples. The Young’s modulus was expressed as the slope in the linear part from stress-strain curves. At least five samples per polymer were analyzed. Hydrophilicity and biodegradation analysis in vitro. The hydrophilicity was expressed as the water contact angle test through a goniometer and imaging system (SL200KB, Kino). The water contact angle on sample was captured and measured after 1 min dropping. At least six different positions on the sample were measured. The in vitro degradation of sample was carried out in a phosphate buffer solution (PBS, pH=7.4) for 28 days at 37℃. Briefly, the PCE film with a size of 1 cm×1 cm and weight of W0 was soaking in PBS in a centrifuge tube and the solution was changed every the other day. At the predetermined time point, the sample was removed and dried to be a balanced weight (Wt). The weight loss of samples was calculated through an equation 1: Weight loss%=(W0-Wt)×100/W0

(1)

Intrinsical photoluminescent ability of PCE elastomers. The spectrophotometer (F-4500, Hitachi, 150 W xenon lamp) was used to evaluate the photoluminescent spectra of PCE elastomers. The excitation and emission slit width were 2.5 nm. The fluorescent images of PCE were captured after excitation at 365 nm using a UV lamp. The photoluminescent quantum yield was performed on a fluorescent spectrometer (F-7000, Hitachi) using an integrating sphere method. The photoluminescent stability of PCE film was determined by continuous illumination for 150 min at 365 nm. Antibacterial activity evaluations in vitro. The broad-spectrum antimicrobial ability of PCE elastomers



was performed using S. aureus (ATCC 6538), E. coli (ATCC 8739), P. aeruginosa (ATCC 9027), E. faecalis (ATCC 29212). Briefly, various bacteria were cultivated in Mueller-Hinton broth (MHB) at 37 ℃ with shaking at 200 rpm. Then, the mid log phase bacteria medium with different concentrations of 1×105~1 1 ×107 CFU/mL (100 µL) was seeded on PCE film (1 cm in diameter). After 24 h incubation at 37 ℃, the different bacteria were removed, diluted for 10-fold, cultured on a tryptic soy agar (Sigma) for another 24 h. The live bacteria colony was captured and the bacteria viability was calculated through counting the colony. At least five species per sample were measured in the antibacterial experiment. In vitro hemocompatibility and cellular biocompatibility investigations. The hemocompatibility of samples was determined by testing the hemolysis using fresh rat erythrocyte cells (ECs). The ECs were collected through centrifugation and rinse by PBS for three times. Fresh packed ECs were dispersed in Tris buffer to form a solution of 5% v/v. Then, various samples (200 mg) were immersed in the ECs solution (1 mL) for 1 h at 37 ℃ with shaking (100 rpm), followed by the centrifuge of suspension at 500 g for 3 min. Then, to test the release of hemoglobin with an absorbance at 540 nm, the the Drabkin’s reagent was mixed with supernatant (10:1 at volume). The absorbance at 540 nm for the supernatants was obtained using a microplate reader (SpectraMax i3, Molecular Devices). The hemolysis percentage of samples was analyzed by equation 2. The ECs incubated with 0.1% Triton x-100 and PBS was used as the negative and positive control respectively. Hemolysis ሺ%ሻ =

Ap ି Ab At ି Ab

×100%

(2)

where Ap, Ab and At were the absorbance values of samples, negative control and positive control, respectively. To investigate the cytotoxicity of PCE elastomers, mouse myoblast (C2C12) was used (Cell Bank, Chinese Academy of Sciences). Before cell culture, the PCE film (1 cm in diameter) was sterilized by 75%


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ethanol for 30 min. The C2C12 cells were seeded on sample with a density of 3000 cells per well (24-well plate). After cultured for 1, 3 and 5 days in Dulbecco's modified Eagles medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37℃, 5% CO2, the samples with cells were washed by PBS for 3 times, followed by incubation with 10% v/v Alamar blue fluorescent dye for 4 hours. The live cells fluorescent intensity (cell viability) was determined through a microreader at 530/600 nm (SpectraMax, Molecular Devices). At least five species per sample were measured. The live-dead staining kit (Invitrogen) was employed to evaluate the live and dead cells on sample after incubation for 24 h. The cell fluorescence was observed by the microscope (IX53, Olympus). Biodegradation, bioimaging and histology evaluations in vivo. The in vivo biodegradation of samples was evaluated by implanting PCE film (size: 1 cm in diameter) at the subcutaneous tissue of Sprague Dawley rat (SD rat). After 8 weeks implantation, the weight loss was calculated according to the equation 1. At least three species per sample were measured to obtain the mean value and standard deviation. To analyze the histology change, the epidermal tissues with samples were fixed by 4% formaldehyde for 24 h. After the paraffin embedding, the samples were sectioned, stained through hematoxylin-eosin (H&E) method and observed under a light microscope. The experimental process was approved by the Animal Ethical Committee of the Academic Medical Center from Xi’an Jiaotong University. Antibacterial activity evaluations in vivo. The rodent subcutaneous infection model was used to evaluate the in vivo antibacterial activity of PCE elastomers. The animal experimental protocol and process was approved by the Animal Ethical Committee at Xi’an Jiaotong University. The female rats (7-8 weeks old, 180-220 g) were acclimated for 7 days before surgical infection. Before implantation, the PCE samples (1 cm in diameter, n=6) were sterilized and seeded by 20 µL inoculums of S. aureus (1×108 CFU/mL). After 10 min incubation, the PCE films were ready for implantation. The rats were anesthetized (10%



chloral hydrate), sterilized with 75% ethanol, and 1 cm incision was carried out on the left and right back in which the PCE 0 and PCE 1.0 film was implanted. After sutured, the rats were housed in ventilated cages for further 6 days. At the time point, the samples were taken out, washed by sterilized PBS, followed by sonication for 10 min to detach the bacteria on sample. The removed bacteria were diluted for 10-fold and cultured on the tryptic soy agar plate for 24 h. The bacteria viability and growth was determined using the similar method with the in vitro experiment. At least three rats per sample were performed. Statistical analysis. All data are shown as the mean ±SD. The statistical Analysis was performed through a Statistical Program for Social Science (SPSS). The significant difference analysis was performed by the student’s T-test and analysis of variance (ANOVA). The statistical difference was significantly when the p-value was below 0.05 and 0.01. ASSOCIATED CONTENT Supporting Information Molecular weight and reaction conditions, fatigue mechanical test, photoluminescent spectra, fibrous capsule thickness, fluorescent images of cells. AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] *E-mail: [email protected] Author Contributions 1

These authors (Li F and Y Su) contributed equally to this work.

Notes The authors declare no competing financial interest.


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ACKNOWLEDGMENTS We acknowledge the valuable comments of potential reviewers. This work was supported by China Postdoctoral Science Foundation (Grant No. 2017M613148), State Key Laboratory for Mechanical Behavior of Materials (Grant No 20161801), National Natural Science Foundation of China (Grant No. 51502237). REFERENCES 1. Yin, J.; Luan, S. Opportunities and Challenges for the Development of Polymer-based Biomaterials and Medical devices. Regenerative Biomaterials, 2016, 3(2),129-135, DOI:10.1093/rb/rbw008 2. Nasution,

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Figure captions


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Figure 1. Synthesis and characterizations of multifunctional and elastomeric PCE biomedical implants. (A) Synthetic process of PCE prepolymer and elastomers; (B) 1H NMR spectra of PCE prepolymer; (C) FTIR spectra of various samples. EDC:1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, NHS: N-Hydroxysuccinimide. Figure 2. Highly elastomeric mechanical properties and controlled biodegradation evaluations. (A) Photographs of PCE 1.0 elastomers after stretched and recovery; (B) Representative tensile stress-strain curves of various samples; (C) Highly elastomeric mechanical properties of samples compared with native soft tissues; (D) Water contact angle test showing the hydrophilicity of samples; (E) In vitro weight loss of samples after soaking in PBS (7.4) for 28 days; (F) Exponential fitting curves of weight loss process demonstrating their linear degradation process during first 24 days. Figure 3. Stable photoluminescent properties and degradation profiles in vivo of PCE elastomers. (A) Photoluminescent pictures of samples under a UV lamp at 365 nm; (B) Photoluminescent emission spectrum excited at 365 nm (inset: fluorescent image); (C) Photoluminescent emission spectrum excited at 365 nm after stored at room temperature for 6 months; (D) Photoluminescent stability analysis through a continual illumination of 150 min at 365 nm; (E) Comparison of in vivo weight loss and in vivo fluorescent loss of PCE 2.0; (F) In vivo fluorescent images of representative mice with a subcutaneously implanted PCE 0.5 and PCE 2.0 film at different time points. Figure 4. Highly efficient broad spectrum antimicrobial activity evaluations in vitro and in vivo. (A) Broad spectrum antimicrobial efficiency of samples after 24 h culture with different microbial concentrations (*p

Biodegradable, Biomimetic Elastomeric, Photoluminescent, and Broad

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