Facile Surface Multi-Functionalization of Biomedical Catheters with

Subscriber access provided by MT SINAI SCH OF MED

Article

Facile Surface Multi-Functionalization of Biomedical Catheters with DualMicrocrystalline Broad-spectrum Antibacterial Drugs and Antifouling Poly(ethylene glycol) for Effective Inhibition of Bacterial Infections Manman Yu, Xuejia Ding, Yiwen Zhu, Shuangmei Wu, Xiaokang Ding, Yang Li, Bingran Yu, and Fu-Jian Xu ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.9b00049 • Publication Date (Web): 31 Jan 2019 Downloaded from http://pubs.acs.org on February 3, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Bio Materials

Facile Surface Multi-Functionalization of Biomedical Catheters with Dual-Microcrystalline Broad-spectrum Antibacterial Drugs and Antifouling Poly(ethylene glycol) for Effective Inhibition of Bacterial Infections Manman Yu, a,b,c† Xuejia Ding, a,b,c† Yiwen Zhu, a,b,c Shuangmei Wu, a,b,c Xiaokang Ding, a,b,c Yang Li, a,b,c Bingran Yu, a,b,c, * and Fu-Jian Xu a,b,c * aState

Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, Beijing 100029 China b Key Lab of Biomedical Materials of Natural Macromolecules (Beijing University of Chemical Technology), Ministry of Education, Beijing 100029 China cBeijing Laboratory of Biomedical Materials, Beijing Advanced Innovation Center for Soft Matter Science and Engineering, Beijing University of Chemical Technology, Beijing 100029 China †Both

authors contributed equally to this work. *Corresponding authors. Tel.: 8610-64421243 E-mail address: [email protected] (F.J. Xu); [email protected] (B. Yu)

1

ACS Paragon Plus Environment

ACS Applied Bio Materials 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT With the development of biomedical materials, the widespread use of implantable medical devices such as biomedical catheters have saved lives and improved therapeutic outcomes in the clinic. Biomedical catheters (BCs) have the ability to connect the body inside and outside, which are widely used in the clinical sites for fluid discharging, blood indwelling, mechanical ventilating and so on. However, catheterrelated infections (CRIs) are common nosocomial infections with high morbidity and mortality. The pathogens in the urinary tract, blood, and lung tissue carried by BCs may be the direct cause of CRIs, and the bacterial biofilm on the surface of BCs provides a notable source of persistent diseases. Microcrystalline sulfamethoxazole (SMZ) and trimethoprim (TMP) were prepared in this study to increase both the specific surface area and water-solubility of antibacterial drugs, as well as to enhance the antibacterial and antifouling effects on the surface of BCs. Then, as-prepared drugs and the excellent antifouling agent polyethylene glycol (PEG) were used for the functionalization of BCs. The result indicated that the sizes of microcrystalline SMZ and TMP were 0.5-3 μm, 1-5 μm, respectively. The coating of BC-PEG-drugs exhibited excellent antibacterial efficacy in culture as well as preeminent antibacterial and antifouling abilities on the surface of BCs toward Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli). Moreover, the BC-PEG-drugs groups exhibited outstanding antibacterial and antifouling abilities in vivo by an animal infection model with S. aureus. This study offers a simple and effective approach for the synthesis of antibacterial and antifouling coatings that consist of microcrystalline drugs, with promising clinical applicability. KEYWORDS: microcrystalline drugs, infection, antibacterial, antifouling, multifunctionalization

2


Page 2 of 32

Page 3 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1. INTRODUCTION Bacterial infection remains a main obstacle to patients’ recovery during the implantation of medical devices despite strict aseptic procedures in surgery.1-2 Among these implants, biomedical catheters (BCs) are widely used in the urinary system, for blood indwelling and mechanical ventilating. However, most of available BCs are prone to microbial colonization, which is likely due to their hydrophobic characteristics.3 Therefore, catheter-related infections (CRIs) are the common and severe types of nosocomial infections.4 BCs inserted into patients may provide direct access for pathogens into urinary tracts and lung tissues, and a bacterial biofilm may easily form on the surface of BCs in a short time, which causes the deterioration of infections and microbial persistence.5-7 Therefore, the solutions of CRIs mainly focused on eradicating bacteria quickly and enhancing the antimicrobial and antifouling abilities of BC surfaces. Both sulfamethoxazole (SMZ) and trimethoprim (TMP) have been clinically used for various infections since 1960s.8 Both SMZ and TMP exhibit potent and broadspectrum antibacterial activities towards both Gram-negative and Gram-positive bacteria, and their combined use can reduce drug resistance.9-11 However, the large sizes of drugs in water block their widespread application.12-14 In addition, many CRIs such as lungs infection are hard to be treated via directly injecting antibiotics. Intravenous injection may lead to the toxicity of other normal tissues and cells. Therefore, the surface functionalization of BCs is central for the treatment of the infections. There are some reported antibacterial coatings with antibiotics,15 antimicrobial peptides,16 3



silver,17,18 polyzwitterions,19 and enzymes.20 However, these surface coatings may be inhibited for effective applications by antimicrobial resistance,21 high cost of synthesis,22 and denaturation in extreme conditions.23 Furthermore, it would be difficult to inhibited the biofilm by a monofunctional antibacterial coating.24 Thus, it is of great urgency to synthesize multi-functionalized coating with broad-spectrum antibacterial and antifouling functionalities.25-27 Herein, we develop a facile and effective strategy for constructing antibacterial and antifouling coatings by co-deposition of dual-microcrystalline drugs and one antifouling polyethylene glycol (PEG) agent as shown in Figure 1. Pristine SMZ and TMP have large sizes in water and block their widespread application. Microcrystalline form is a kind of pharmaceutical preparation to improve the absorption of drugs as reported.28 The crystalline powder with micrometer scales are regard as microcrystalline.29 Through the physical deposition, these microcrystalline drugs were deposited on the surface of BCs to rapidly form one antibacterial coating, which can eradicate bacteria quickly. The combined use of SMZ and TMP can effectively reduce the antimicrobial resistance. At the meantime, antifouling PEG was introduced into the system to provide a hydrophilic environment on the surface, which can reduce both cells adsorption and bacteria adhesion at the material interface.30-33 The antibacterial and antifouling abilities of the microcrystalline-drug-modified BCs were studied in vitro and in vivo.

4


Page 4 of 32

Page 5 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


2. EXPERIMENTAL SECTION 2.1. Materials Sulfamethoxazole (SMZ; > 98.0%) and trimethoprim (TMP; > 98.0%) were obtained from Tokyo Chemical Industry (TCI). Poly(acrylic acid) was purchased from Acros Organics (Geel, Belgium). mPEG-Amine (Mn = 5000 Da) was purchased from J&K Scientific LTD (Beijing, China). Ethanol (A.R. grade) was purchased from the Beijing Chemical Works (China). The pathological glass slides were purchased from Citotest (Jiangsu, China). Tryptone and yeast extract were purchased from Oxoid (UK). The strains of Escherichia coli (E. coli, ATCC 25922) and Staphylococcus aureus (S. aureus, ATCC 25923) were purchased from Promega (Madison, U.S.A.). The Live/Dead BacLight bacterial viability kit (L7012) was purchased from Invitrogen (Life Technologies, U.S.A.). The lysogeny broth (LB) medium and deionized water were autoclaved (120 °C, 20 min) before use. The BCs and PVC slides (Mw = 50000~110000 Da) were purchased from Henan Tuoren Group (Henan, China). 2.2. Microcrystallization of SMZ and TMP The preparation of microcrystalline drugs was improved based on the crystallization. Microcrystalline SMZ and TMP drugs were prepared by dissolving 100 mg SMZ or TMP in 1 mL of ethanol. Then, 10 mL of hot water (80 °C) and 20 mg poly(acrylic acid) were added into the SMZ suspension, allowing the reaction to remain at room temperature under constant stirring for 6 h. The only difference between the preparation of microcrystalline TMP and SMZ was that the TMP suspension was prepared without poly(acrylic acid). The samples were collected by centrifugation at 10000 rpm for 10 min. Then, the precipitations were suspended with deionized water and centrifuged again for three times. Finally, the products were gained by vacuum freeze-dried and 5



Page 6 of 32

stored at 4 °C. 2.3. Characterization of Microcrystalline Drugs The particle sizes of the microcrystalline drugs were measured by a Zetasizer nano (ZS) equipped with a laser of wavelength 633 nm at a 173° scattering angle (Malvern Instruments, Southborough, MA, USA). Scanning electron microscope (SEM, JEOL, JSM-7500F) was used to study the morphologies of both microcrystalline drugs and pristine drugs. The melting enthalpies and temperatures of microcrystalline drugs and pristine drugs were measured by DSC Q20 V24.11 at a heating rate of 10 °C /min. 2.4. Antibacterial Activity in Solution The antibacterial activity of the microcrystalline and pristine drugs (SMZ:TMP = 5:1 W/W) in solution against Gram-negative bacteria (E. coli) and Gram-positive bacteria (S. aureus) were evaluated by inhibiting efficiency and minimum inhibitory concentration (MIC), which was described in detail in the Supporting Information. 2.5. Preparation and Characterization of Microcrystalline Drugs Coatings of BCs Poly(vinyl chloride) (PVC) substrates were chosen as a typical substitute for BCs. PVC is more commonly used as catheter material due to its high tensile strength, softness, pliability, inherently chemical resistance, biocompatibility, and ability to meet flow requirements. As for the preparation of coatings, BCs (Φout = 9.93 mm; Φin = 8.73 mm) were first cut into fragments with a height of 2 mm.

To characterize the surface

of BCs, the PVC slides were cut into squares of 5 mm × 5 mm. Then, the BCs and PVC slides were coated with a layer of polydopamine (PDA) following previously published procedures.29 The BCs and PVC slides modified with polydopamin (PDA) were transferred into an aqueous suspension containing 1 mL microcrystalline SMZ and TMP (4 mg/mL and 0.8 mg/mL) and PEG (2 mg/mL), microcrystalline SMZ and TMP (4 mg/mL and 0.8 mg/mL); PEG (2 mg/mL) in a 48-well plate, respectively. After 6


Page 7 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


incubation at room temperature for 12 h, all BCs and PVC slides with different coatings (namely BC-PEG-drugs, BC-drugs, and BC-PEG) were dried at room temperature. Pristine BCs and PVC slides with pristine drugs which had the similar amount of drugs as that of BC-drugs group were regarded as the Control Drugs groups. Pristine SMZ (10 mg/mL) and Pristine SMZ (5 mg/mL) dissolved in methanol were slowly and carefully dropped onto the surfaces of pristine BCs and PVC slides. Surface characterization of all the BCs and PVC slides was studied via SEM, X-ray photoelectron spectroscopy (XPS), and static water contact angles (WCAs). The details are described in the Supporting Information. 2.6. Loading and Releasing of Microcrystalline Drugs Loading and releasing of microcrystalline drugs were measured through high performance liquid chromatography (HPLC, Shimadazu, Japan). BC-PEG-drugs and BC-drugs were soaked in 5 mL mobile phase (water: acetonitrile: ethylene diamine: acetic acid = 7: 3: 0.2: 0.1, pH = 5.8). The UV wavelength of 240 nm was applied to analyze drugs on a C18 column at a flow rate of 1 mL/min. BC-PEG-drugs, BC-drugs, and Control Drugs groups were added to 5 mL acetic acid buffer (pH = 5.5). Supernatant liquid (0.5 mL) was obtained for measuring the released drugs from BCPEG-drugs, BC-drugs, and Control Drugs at specific time points. Then, the same volume of fresh acetic acid buffer was supplied in the release medium. 2.7. Antibacterial and Antifouling Abilities of Modified BCs The antibacterial and antifouling activities were studied using zones of inhibition assay and live/dead fluorescence staining assay. The Supporting Information showed the procedures in detailed. 2.8. Inhibition of Biofilm Formation All modified BCs were mixed with E. coli and S. aureus suspensions in LB medium 7



(500 μL, 1 × 108 cfu/mL), respectively. After incubation at 37 °C for 7 days to allow bioﬁlm formation, all BCs were washed with normal saline solution and stained with SYTO 9 (6 μmol/L) and propidium iodide (PI, 30 μmol/L) for 15 min. Biofilms were exposed to modified BCs and examined with confocal laser scanning microscopy (CLSM) using an oil immersed 63× objective lens. 2.9. Hemocompatibility of the Antibacterial Coatings The red blood cells (RBCs) were obtained from the fresh blood of mice by washing and centrifuging (2000 rpm, 10 min) for three times. To investigate the hemocompatibility of the coatings, modified BCs were immersed in 1 mL PBS for 24 h, and then the soaking solutions were incubated with 2% v/v of RBCs for 3 h. The positive and negative controls were the RBCs incubated with deionized water and PBS solution, respectively. 2.10. In Vivo Anti-Infection Assay Female BALB/c mice (8-week-old, 20 g) were used for the in vivo anti-infection assay. All protocols involving mice comply with the guidelines described in the Association for Assessment and Accreditation of Laboratory Animal Care.35 After adaption for one week, all mice were randomly divided into four groups (four mice per group): (1) with infection BC-PEG-drugs; (2) with infection BC-drugs; (3) with infection Pristine-BC (control (+)); (4) with pristine BC (control (-)). The Supporting Information showed the procedures in detailed. 2.11. Statistical Analysis Each experiment was repeated at least three times, and data are shown as means ± standard deviation. Statistical significance (*p value < 0.05) was evaluated with the student t-test when only two groups were compared. If more than two groups were 8


Page 8 of 32

Page 9 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


compared, evaluation of significance as performed using one-way analysis of variance (ANOVA) followed by Bonferroni’s post hoc test. In all tests, statistical significance was set at p < 0.05.

9



3. RESULTS AND DISCUSSION 3.1. Synthesis and Characterization of Microcrystalline Drugs The size and size distribution of microcrystalline SMZ and TMP were characterized using SEM and dynamic light scattering (DLS). Pristine SMZ and TMP were irregular and large size as shown in Figure 2a. In contrast, both of microcrystalline of SMZ and TMP drugs exhibited more regular and morphology smaller size. Microcrystalline SMZ and TMP had good dispersion in water as shown in the Figure 2b. The particle sizes of microcrystalline SMZ ranged from 0.5 to 3 µm and that of microcrystalline TMP ranged from 1 to 5 µm. As showed in the Figure S1, the melting temperatures of microcrystalline (or pristine) SMZ and TMP are 170.84 °C (or 170.96 °C) and 202.28°C (or 202.26 °C), respectively. The melting enthalpies of microcrystalline (or pristine) SMZ and TMP are 117.0 J/g (or 127.5 J/g) and 133.1 J/g (or 173.5 J/g), respectively. The melting enthalpies of drugs were reduced with the sizes of crystal decreased. The above data proved the successful preparation of the microcrystalline drugs. 3.2. Antibacterial Activity in Solution Both drugs exhibit potent antibacterial activities towards Gram-negative (S. aureus) and Gram-positive (E. coli) bacteria, and their combined use can reduce drug resistance.10 In terms of economy and effect, the optimum ratio of SMZ:TMP is 5:1 in clinical.8 The antimicrobial activities of the microcrystalline and pristine drugs (SMZ:TMP=5:1) were studied in solution (Figure 2c and Figure S2). The minimum inhibitory concentration (MIC) values of both microcrystalline and pristine drugs were 512 ng mL-1 for S. aureus and 4096 ng mL-1 for E. coli. Microcrystalline drugs still showed very low MIC for both S. aureus and E. coli, which indicated that 10


Page 10 of 32

Page 11 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


microcrystalline drugs exhibited excellent antibacterial activity. 3.3. Physical Characterization of the Coatings The morphology and size of microcrystalline drugs were not affected during the physical deposition process (Figure 3). Compared with the coating of Control Drugs, the size of microcrystalline drugs of BC-PEG-Drugs and BC-Drugs was much smaller. The surface of Pristine BC was smooth and became rough after modification with polydopamine (PDA) and PDA-PEG (Figure 3 and Figure S3, Supporting Information). The hydrophilicity of the modified BCs was measured by water contact angles (WCAs). The contact angle of pristine BC and PDA modified BC were 109 ± 1° and 79 ± 2°, respectively. When modified with PEG, the contact angle of BC-PEG significantly decreased to 27 ± 8° (Figure S3), indicating successful modification. Both of the surfaces of BC-Drugs and BC-PEG-Drugs showed contact angles around 60°, while the contact angles of Control Drugs were 78 ± 5°, which may attribute to the bigger size of drugs. All groups were characterized by XPS. The elemental percentages and XPS spectra of BC-PEG-Drugs, BC-Drugs, BC-PEG, BC-PDA, Control Drugs and Pristine BC were showed in Table S1 and Figure S4. The O 1s peak of Pristine BC was due to the plasticizers (epoxy soybean oil) of PVC. A high N 1s peak of BC-PDA was raised compared to Pristine BC, indicating the successful decoration with PDA. BC-PEG had the highest percentage of C-O peak, due to the C-O bond of PEG. A new peak of P 2s was observed on the surfaces of BC-PEG-Drugs and BC-Drugs, confirming successful deposition of microcrystalline SMZ and TMP. Furthermore, the C element of BC-PEGDrugs showed higher percentage of the C-O bond than BC-Drugs, indicating that PEG were successfully anchored on the surface. All these results confirm the successful modification of coatings by different materials on BCs. 11



3.4. Loading and Releasing of Microcrystalline Drugs To measure the drug loading and drug releasing ratio of modified BCs, we matched a series of gradient solution of SMZ and TMP to obtain the standard curve (Figure S5). The SMZ loading of BC-PEG-Drugs and BC-Drugs were approximately 0.8 μg/mm2 and 1.0 μg/mm2, respectively. The TMP loading of both BC-PEG-Drugs and BC-Drugs were approximately 0.1 μg/mm2 (Figure S5). The pH value of the infected site would reduce to 6.0 or below due to the formation of a great quantity of H+.31 In the case of simulated infection (pH = 5.5), the drug releasing ratios of SMZ and TMP from BCPEG-Drugs and BC-Drugs are shown in Figure 4. After immersion for 8 h the total releasing ratio of microcrystalline drugs was about 100%, which is significantly higher than for the Control Drugs. Such excellent releasing efficiently is mainly because microcrystalline drugs with smaller size facilitate its solubilization, which will endow BC-drugs and BC-PEG-drugs with good antibacterial capacity. 3.5. Antibacterial and Antifouling Abilities of Modified BCs The antibacterial activities of drugs released from BC-PEG-Drugs, BC-Drugs, Control Drugs, BC-PEG, BC-PDA, and Pristine BC were first evaluated via the zones of inhibition assay. S. aureus and E. coli colonies were observed around BC-PEG, BCPDA, and Pristine BC, indicating that those BCs cannot inhibit the growth of S. aureus and E. coli (Figure 5). In contrast, the growths of both S. aureus and E. coli were significantly inhibited around BC-PEG-Drugs, BC-Drugs, and Control Drugs. The zone of inhibition of the Control Drugs was significantly less than those of BC-Drugs, and BC-PEG-Drugs based on one-way analysis of variance (ANOVA) followed by Bonferroni’s post hoc test, which was consistent with Figure 4. 12


Page 12 of 32

Page 13 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


The live/dead fluorescence staining assay performed in S. aureus and E. coli was shown in Figure 6. The green fluorescence on Pristine BC, BC-PDA, and BC-PEG, showed that most S. aureus and E. coli bacterial were alive. In contrast, red fluorescence was observed on the BC-PEG-Drugs and BC-Drugs, because the coatings killed most of the S. aureus and E. coli cells on the surface. In order to explore the fouling performance of the PEG, we selected randomly the different positions of modified surfaces, counted the number of bacteria on each CLSM image, and obtained the averages as shown in Figure S8. A few S. aureus and E. coli remained on the surface of BC-PEG and BC-PEG-Drugs, indicating good antifouling abilities of BC-PEG and BC-PEG-Drugs due to PEG moieties. After incubation with the BCs, the viability of S. aureus and E. coli in the culture medium was examined to address the antibacterial behavior of the released drugs. The live/dead fluorescence staining assay of solutions is shown in Figure 6, where many dead S. aureus and E. coli cells were observed and the number of bacteria decreased obviously of the BC-PEG-Drugs and BC-Drugs. The optical densities (OD600) of the E. coli and S. aureus cultures are shown in Figure S7, and the culture without materials was used as control group. After incubating BC-PEG-Drugs and BC-Drugs in the culture, around 80% of E. coli and S. aureus cells were killed. 3.6. Inhibition of Biofilm Formation Biofilm inhibition is one of the most important properties that should be evaluated for the treatment of CRIs. The surface of Pristine BC was covered by live biofilm, and the thickness of S. aureus and E. coli were 3 μm and 6 μm (Figure 7)., respectively. 13



Significantly less bacteria were observed on the surface of BC-PEG and BC-PEGDrugs, which reflected the antifouling of PEG. In addition, BC-Drugs displayed more dead cells compared to BC-PEG-Drugs. In summary, the BC-PEG-Drugs exhibited outstanding performance in inhibiting formation of biofilm. 3.7. Hemocompatibility of Antibacterial Coatings Good biocompatibility is critical for biomedical materials, which is measured by hemolysis assay.35 As shown in Figure 8b, hemolysis ratios were less than 5% when incubated with the soaking solution of BC-PEG-Drugs, BC-Drugs, BC-PEG, BC-PDA, and Pristine BC, which were similar to that of PBS, indicating good biocompatibility of our materials. Figure 8a showed that the supernatant of RBCs suspensions was clear after incubation with all coatings, which was consistent with the results shown in Figure 8b. The morphology of RBCs was observed by CLSM (Figure 8c), in which complete cell morphology indicated the safety of our materials. 3.8. In Vivo Anti-Infection Assay The antibacterial and antifouling properties of coatings in vivo were investigated by an animal infection model as previously reports.34-35 In our work, the Pristine BC with infection was considered to be the control (+) group, and the Pristine BC without infection was the control (-) group. Firstly, macroscopic assessment of the wound inflammation was performed. The mice were observed for 14 days after surgery. As shown in Figure 9, wound inflammation occurred after 4 days. The control (+) group showed much stronger inflammatory reaction than the BC-PEG-Drugs, BC-Drugs, and control (-) groups, accompanied by wound redness and maturation. This situation continued for 14 days. The wounds of the BC-PEG-Drugs and BC-Drugs groups healed on day 7, which was similar to control (-) group. At the end of the observations at 1 day, 4 days, 7 days, and 14 days, the number of bacteria in each group (Figure 9b and 14


Page 14 of 32

Page 15 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


9c) was counted. After implantation for 1 day, the number of bacteria of the BC-PEGDrugs and BC-Drugs groups was significantly lower than the control (+) group. After 4, 7 and 14 days, reduced number of bacteria on the LB-agar plate was observed in the BC-PEG-Drug and BC-Drug groups. The surfaces of the BC-Drugs and BC-PEG-Drugs groups adhered less bacteria than the control (+) group after 1 day by CLSM (Figure 9a). Some cells and debris instead of colony can be seen in the control (-), BC-Drugs, and BC-PEG-Drugs groups after 4, 7, and 14 days, which were characterized in Figure 9a by SEM. The results suggest the superior antifouling and antibacterial properties of BC-PEG-Drugs. The DNA sequence of the bacteria grown in mice was consistent with S. aureus in the NCBI database. (Figure 9d and Figure S9). The pathological sections of mice were taken, and eosin (H&E) staining was conducted to detect the inflammatory response caused by the injected bacteria (Figure 10). For mice skin and muscle, neutrophilic infiltration occurred on the tissue of control (+) group after 1 day, indicating that these tissues had a significant inflammatory response. Neutrophil also appeared in the control (-) group due to the implantation procedure of BCs. In comparison, the amounts of neutrophil in the BC-PEG-Drugs and BC-Drugs groups were lower, indicating a milder inflammatory response. The neutrophil infiltration of the control (-) group decreased, and control (+) group exhibited disintegrated neutrophil with severe infiltration after 7 days. Moreover, the formation of new vessels and granulomatous inflammation of the BC-PEG-Drugs, BC-Drugs and control (-) groups indicated a healing process. After 14 days, the control (+) group showed a more severe inflammatory reaction, accompanied by more neutrophil and 15



fibrinous inflammation in the tissue. In conclusion, the anti-infection assay indicated that BC-PEG-Drugs had significant antibacterial and antifouling abilities on the surface, and the drugs released from the modified BCs could eradicate the bacteria rapidly in vivo.

16


Page 16 of 32

Page 17 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


4. CONCLUSIONS In summary, we successfully fabricated efficient antibacterial and antifouling coatings on BCs through a facile deposition approach. The dual-microcrystalline of drugs and antifouling PEG were simultaneously coated on BCs. As prepared BCs showed good drugs loading capacity and excellent releasing efficacy. The in vitro and in vivo anti-infection assays proved that BC-PEG-Drugs owned outstanding antibacterial and antifouling abilities. Moreover, the drugs released from the modified BCs could eradicate bacteria rapidly. This work with promising clinical applicability would provide a simple and effective approach to surface functionalization of BCs to reduce CRIs.

SUPPORTING INFORMATION Detailed experimental methods, SEM images, XPS spectra, water contact angles, drugs loading of BC-PEG-Drugs and BC-Drugs, HPLC data and gene sequence of bacteria are available. ACKNOWLEDGEMENTS This work was supported by National Key Research and Development Program of China (grant numbers 2016YFB1101200), National Natural Science Foundation of China (grant numbers 51733001 and 21875014), Beijing Natural Science Foundation (Grant No. L160004 and 7161001), and Fundamental Research Funds for the Central Universities (PYBZ1814, PYBZ1839 and XK1802-2).

17



REFERENCES [1] Brown E. D.; Wright G. D. Antibacterial Drug Discovery in the Resistance Era. Nature 2016. 529, 336-343. [2] Villanueva, M. E.; González, J. A.; Rodríguez-Castellón, E.; Teves, S.; Copello, G. J. Antimicrobial Surface Functionalization of PVC by a Guanidine based Antimicrobial Polymer. Mat Sci Eng C 2016. 67, 214-220. [3] Siddiq, D. M.; Darouiche, R. O. New Strategies to Prevent Catheter-Associated Urinary Tract Infections. Nat. Rev. Urol. 2012. 9, 305-314. [4] Neoh, K. G.; Li, M.; Kang, E.-T.; Chiong, E.; Tambyah, P. A. Surface Modification Strategies for Combating Catheter-Related Complications: Recent Advances and Challenges. J. Mater. Chem. B 2017. 5, 2045-2067. [5] Xu, K.; Liang, L.; Cui, M.; Han, Y.; Karahan, H. E.; Chow, V. T. K.; C, Xu. ColdChain-Free Storable Hydrogel for Infant-friendly Oral Delivery of Amoxicillin for the Treatment of Pneumococcal Pneumonia. ACS. Appl Mater Interfaces 2017. 9, 1844018449. [6] Ivanova, K.; Fernandes, M. M.; Francesko, A.; Mendoza, E.; Guezguez, J.; Burnet, M.; Tzanov T. Quorum-Quenching and Matrix-Degrading Enzymes in Multilayer Coatings Synergistically Prevent Bacterial Biofilm Formation on Urinary Catheters. ACS Appl. Mater. Interfaces 2015. 7, 49, 27066-27077. [7] Gomes, L. C.; Silva, L. N.; Simões, M.; Melo, L. F.; Mergulhão, F. J. Escherichia coli Adhesion, Biofilm Development and Antibiotic Susceptibility on Biomedical Materials. J. Biomed. Mater. Res. A 2015. 103, 1414-1423, [8] Bushby, S. R. M.; Hitchings, G. H. Trimethoprim, a Sulfonamide Potentiator. J Pharmacology Chemotherapy. 1968. 33, 72-90. [9] Vo, A.; Wassner, C.; Ruggiero, N. N.; Mukherji, R.; Cohen, H. Probable Qtc Prolongation and Bradycardia after Sulfamethoxazole/Trimethoprim Administration. Crit Care Med. 2018. 46, 433. [10] Masters, P. A.; O'Bryan, T. A.; Zurlo, J.; Miller, D. Q.; Joshi, N. TrimethoprimSulfamethoxazole Revisited. Arch. Intern. Med. 2003. 163. 402-410. 18


Page 18 of 32

Page 19 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


[11] Hawser, S.; Lociuro, S. K. Dihydrofolate Reductase Inhibitors as Antibacterial Agents. Biochem. Pharmacol. 2006. 71, 941. [12] Patton, J. S.; Byron, P. R. Inhaling Medicines: Delivering Drugs to the Body through the Lungs. Nat. Rev. Drug Discovery 2007. 6, 67-74. [13] Sturm, R. Deposition and Cellular Interaction of Cancer-Inducing Particles in the Human Respiratory Tract: Theoretical Approaches and Experimental Date. Thorac Cancer 2010. 1, 141-152. [14] Yang, X.; Wu, S.; Li, Y.; Huang, Y.; Lin, J.; Chang, D. Integration of an AntiTumor Drug into Nanocrystalline Assemblies for Sustained Drug Release. Chem. Sci. 2015. 6, 1650-1654. [15] Campoccia, D.; Montanaro, L.; Arciola, C. R. A Review of the Biomaterials Technologies for Infection-Resistant Surfaces. Biomaterial 2013. 8533–8554. [16] Mishra, B.; Basu, A.; Chua, R. N. R. Y. R.; Saravanan, P. A.; Tambyah, B. Ho.; Chang, M. W.; Leong, S. S. Site Specific Immobilization of a Potent Antimicrobial Peptide onto Silicone Catheters: Evaluation Against Urinary Tract Infection Pathogens. J. Mater. Chem. B 2014. 1706. [17] Lemire, J. A.; Harrison, J. J.; Turner, R. J.; Antimicrobial Activity of Metals: Mechanisms, Molecular Targets and Applications, Nat. Rev. Microbiol. 2013. 11, 371384. [18] Honda, M.; Kawanobe, Y.; Ishii, K.; Konishi, T.; Mizumoto, M.; Kanzawa, N.; Matsumoto, M.; Aizawa, M. In Vitro and in Vivo Antimicrobial Properties of Silver Containing Hydroxyapatite Prepared via Ultrasonic Spray Pyrolysis Route. Mater. Sci. Eng. C 2013. 33, 5000-5018. [19] Green, J.-B. D.; Fulghum, T.; Nordhaus, M. A.; Immobilized Antimicrobial Agents: A Critical Perspective. Chem. Rev. 2009. 109, 5437-5527. [20] Hedstrom, L. Serine Protease Mechanism and Specificity. Chem. Rev. 2002. 102, 4501-4524. [21] Cao, B.; Xiao, F.; Xing, D.; Hu, X. Polyprodrug Antimicrobials: Remarkable Membrane Damage and Concurrent Drug Release to Combat Antibiotic Resistance of 19



Methicillin-Resistant. Staphylococcus aureus. Small 2018, 14, 1802008. [22] Lo, J.; Lange, D.; Chew, B. Ureteral Stents and Foley Catheters-Associated Urinary Tract Infections: The Role of Coatings and Materials in Infection Prevention. Antibiotics. 2014. 3, 87-97. [23] Thallinger, B.; Brandauer, M.; Burger, P.; Sygmund, C.; Ludwig, R.; Ivanova, K.; Kun, J.; Scaini, D.; Burnet, M.; Tzanov, T.; Nyanhongo, G. S.; Guebitz, G. M. Cellobiose Dehydrogenase Functionalized Urinary Catheter as Novel Antibiofilm System. J. Biomed. Mater. Res. B Appl. Biomater. 2016. 104, 1448-1456. [24] Zhi, Z.; Su, Y.; Xi, Y.; Tian, L.; Xu, M.; Wang, Q.; Padidan, S.; Li, P.; Huang, W. Dual-Functional Polyethylene Glycol‑b‑Polyhexanide Surface Coating with in Vitro and in Vivo Antimicrobial and Antifouling Activities. ACS Appl. Mater. Interfaces 2017. 9, 10383-10397. [25] Chen, S.; Yuan, L.; Li, Q.; Li, J.; Zhu, X.; Jiang, Y.; Sha, O.; Yang, X.; Xin, J. H. J.; Wang, Stadler, F. J.; Huang, P. Durable Antibacterial and Nonfouling Cotton Textiles with Enhanced Comfort via Zwitterionic Sulfopropylbetaine Coating Durable Antibacterial and Nonfouling Cotton Textiles with Enhanced Comfort via Zwitterionic Sulfopropylbetaine Coating. Small 2016, 12, 3516-3521. [26] Voo, Z. X. M.; Khan, Q.; Xu, K.; Narayanan, B. W. J.; Ng, R. B.; Ahmad, J. L.; Hedrick, Y.; Yang, Y. Antimicrobial Coatings Against Biofilm Formation: The Unexpected Balance between Antifouling and Bactericidal Behavior. Poly. Chem. 2016. 7, 656-668. [27] Yang, H.; Li, G.; Stansbury, W. J.; Zhu, X.; Wang, X.; Nie, J. Smart Antibacterial Surface Made by Photopolymerization. ACS Appl. Mater. Interfaces 2016, 8, 2804728054. [28] Lockner, D.; Paul. C.; Comparison of A New Microcrystalline Dicoumarol Preparation with Warfarin under Routine Treatment Conditions. Br. J. clin. Pharmac. 1979, 8, 59-64. [29] Mohac, M. L.; Pina, M. F.; Raimi-Abraham, B. T.; Solid Microcrystalline Dispersion Films as A New Strategy to Improve the Dissolution Rate of Poorly Water 20


Page 20 of 32

Page 21 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Soluble Drugs: A Case Study using Olanzapine. International Journal of Pharmaceutics. 2016, 508, 42-50. [30] Peng, L.; Li, C.; Xi, L.; Lin, J.; Liu, H.; Bing, H. Antibacterial Property of a Polyethylene Glycol-Grafted Dental Material. ACS Appl. Mater. Interfaces 2017. 9, 17688-17692. [31] Mizrahi, B.; Khoo, X.; Chiang, H. H.; Sher, K. J.; Feldman, R. G.; Lee, J. J.; Irusta, S.; Kohane, D. S. Long-Lasting Antifouling Coating from Multi-Armed Polymer. Langmuir 2013, 29, 10087-10094 [32] Kim, S.; Gim, T.; Kang, S. M.; Versatile, Tannic Acid-Mediated Surface PEGylation for Marine Antifouling Applications. ACS Appl. Mater. Interfaces 2015, 7, 6412-6416. [33] Zhi, Z.; Su, Y.; Xi, Y.; Tian, L.; Xu, M.; Wang, Q.; Padidan, S.; Li, P.; Huang, W. Dual-Functional Polyethylene Glycol‑b‑polyhexanide Surface Coating with in Vitro and in Vivo Antimicrobial and Antifouling Activities. ACS Appl. Mater. Interfaces 2017, 9, 10383-10397. [34] Keum, H.; Kim, J. Y.; Yu, B.; Yu, S. J.; Kim, J.; Jeon, H.; Lee, D. Y.; Im, S. G.; Jon, S. Prevention of Bacterial Colonization on Catheters by a One-Step Coating Process Involving an Antibiofouling Polymer in Water. ACS Appl. Mater. Interfaces 2017. 9, 19736-19745. [35] Zeng, Q.; Zhu, Y.; Yu, B.; Sun, Y.; Ding, X.; Xu, C.; Wu, Y. W.; Tang, Z.; Xu, F-J. Antimicrobial and Antifouling Polymeric Agents for Surface Functionalization of Medical Implants. Biomacromolecules 2018. 19, 2805-2811.

21



Figure 1. Schematic illustration of the modification route of biomedical catheters and the resultant antibacterial characteristics.

22


Page 22 of 32

Page 23 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 2. (a) SEM images of Pristine SMZ and Pristine TMP in water. (b) SEM images and size distributions of microcrystalline SMZ and microcrystalline TMP. (c) Inhibition efficiencies of the antibacterial microcrystalline SMZ-TMP against S. aureus and E. coli, respectively. The error bars indicate the standard deviation (n = 3).

23



Figure 3. Physical characterizations of the coatings. (a) SEM images, (b) wettability of BC-PEG-Drugs, BC-Drugs, Control Drugs coatings and Pristine BC.

24


Page 24 of 32

Page 25 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 4. (a) SMZ and (b) TMP releasing of BC-PEG-Drugs, BC-Drugs and Control Drugs in acetic acid buffer solution (pH = 5.5) at room temperature with static position. HPLC peaks of the drugs releasing of the BC-PEG-Drugs (c) and (d) BC-Drugs at specific time.

25



Figure 5. Photographs and diameters of the zones of inhibition (ZOIs) of modified BCs showing antibacterial activity of (a) S. aureus and (b) E. coli. The error bars indicate the standard deviation (n = 3).

26


Page 26 of 32

Page 27 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 6. Antibacterial activities of (a) S. aureus and (b) E. coli after 8 h incubation. (a1) and (b1) are fluorescence images of live/dead fluorescence staining assay for the bacteria cells on surface, (a2) and (b2) are fluorescence images of live/dead fluorescence staining assay for the bacteria cells in solution. (scale bar: 25 μm).

27



Figure 7. 3D CLSM images of the bioﬁlms of S. aureus and E. coli on the surfaces of BC-PEG-Drugs, BC-Drugs, BC-PEG, BC-PDA, and Pristine BC after 7-day incubation.

28


Page 28 of 32

Page 29 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 8. (a, b) Hemolysis assay of the soaking solutions of modified BCs. (c) Images of RBCs incubated with soaking solutions. (scale bar: 5 μm). The error bars indicate the standard deviation (n = 3).

29



Figure 9. In vivo anti-infection assay. (a) Wound photographs of different groups of mice after implant experiment, fluorescence images of the surface of the ex vivo BCs after 1, 4, 7, 14 days and SEM images after 14 days. (scale bar: 25 μm). (b) Plate counting photos (scale bar: 2 cm) and (c) the number of viable bacteria in soft tissues around the BCs in the subcutaneous infection model after 1, 4, 7, 14 days. (d) Parts of the 16S rDNA sequencing results of the bacteria (from the infected soft tissues) compared with sequences in NCBI database.

30


Page 30 of 32

Page 31 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 10. Representative images of tissue sections around the functionalized BCs stained with H&E after 1, 4, 7, 14 days. Green dashes represent BCs location (Arrows: black: neutrophils; red: newly formed vessels and granuloma; blue: necrosis). 31



TOC Graphic

32


Page 32 of 32

Facile Surface Multi-Functionalization of Biomedical Catheters with

Recommend Documents