Journal of Materials Science & Technology, 2020, 59(0): 14-25 DOI: 10.1016/j.jmst.2020.05.017

Research Article

Electrostatic assembly functionalization of poly (γ-glutamic acid) for biomedical antibacterial applications

Xiaodan Wanga,b, Hengchong Shia, Haoyu Tangc, Huan Yua,b, Qiuyan Yana, Huawei Yanga, Xu Zhanga, Shifang Luan,a,b,*

a State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, China

b University of Science and Technology of China, Hefei 230026, China

c Institute of Functional Nano & Soft Materials, Soochow University, Suzhou 215123, China

Corresponding authors: *State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, China. E-mail address:sfluan@ciac.ac.cn(S. Luan).

Received: 2020-04-6   Accepted: 2020-04-30   Online: 2020-12-15

Abstract

Poly (γ-glutamic acid) (γ-PGA) has been found widespread applications in biomedical field because of its excellent water solubility, biocompatibility, and bioactivity. Herein, a water-insoluble γ-PGA antibacterial compound is facilely fabricated via one-pot electrostatic assembly of γ-PGA with cationic ethyl lauroyl arginate (ELA). The functionalized γ-PGA compound (γ-PGA-ELA) ethanol solution can facilely produce colorless and transparent coatings on various inorganic, metal, and polymeric substrates, especially for the lumen of slender catheters (length up to 2 m, and inner diameter down to 1 mm). The functionalized γ-PGA coating presents remarkable antibacterial efficacy in vitro and in vivo. In addition, the γ-PGA compound is used as antibacterial additives of polyolefin via melting extrusion, and the as-prepared antibacterial polyolefin demonstrates advantageous antibacterial efficacy. More importantly, the functionalized γ-PGA coating exhibit good hemocompatibility, low cytotoxicity, and satisfactory histocompatibility. The as-proposed γ-PGA compound has a great potential to serve as a safe and multifunctional antibacterial candidate to combat biomedical devices-related infections.

Keywords: Functionalized poly (γ-glutamic acid) compound ; Electrostatic assembly ; Antibacterial coating ; Antibacterial additive ; Catheter-associated infections

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Cite this article

Xiaodan Wang, Hengchong Shi, Haoyu Tang, Huan Yu, Qiuyan Yan, Huawei Yang, Xu Zhang, Shifang Luan. Electrostatic assembly functionalization of poly (γ-glutamic acid) for biomedical antibacterial applications. Journal of Materials Science & Technology[J], 2020, 59(0): 14-25 DOI:10.1016/j.jmst.2020.05.017

1. Introduction

Poly (amino acids) have many attractive properties that make them potentially useful for various in vivo biomedical applications [1,2]. The conjugation of amino acids linked by peptide bonds offers poly (amino acids) biodegradability and biocompatibility [3,4]. Poly (amino acids) can self-assemble into ordered and stable secondary structures, thus they outperform most of synthetic polymers including the most common polyethylene glycol-based materials [[5], [6], [7], [8], [9]]. A variety of active functional groups of poly (amino acids) can be facilely functionalized by biomolecules containing cell-targeted or fluorescent groups to meet different biomedical requirements [[10], [11], [12], [13]]. Among the poly (amino acids), poly (glutamic acid) (PGA) is particularly attractive because of its ease of chemical modification and unique secondary structure responds to changes of pH, which has been widely used in medical fields, such as a drug carrier [14,15], injectable hydrogel [[16], [17], [18], [19],5,8], wound dressing [20] and antibacterial material [9,21].

As for most of the biomedical materials and the related medical devices, they inevitably contact with the outside environment during their practical use, thus posing a risk of bacterial infections [[22], [23], [24]]. Despite aseptic procedures and prophylactic systemic antibiotic therapy, bacterial infections on biomedical implants and devices including catheters, artificial prosthetics, and aortic grafts remain one of the most serious complications in hospital [[25], [26], [27]]. The formation of biofilm increases the tolerance of bacteria, which may lead to the development of bacterial resistance, making the treatment process complicated and difficult [28]. Extensive efforts have been devoted to offering viable options to combat bacterial infections, and the surface coating strategy has been considered as an effective approach [29,30]. The construction of anti-infective coatings can be achieved by multiple methods [31]. Immobilization antibacterial agents on the medical devices through the methods of physical adsorption [32], layer-by-layer self-assembly [33,34], mussel adhesion [35], electrostatic assembly [36], and surface nanostructures (e.g. growing nanostructured TiO2 on Ti implants) [37,38] have all received satisfactory antibacterial results. However, several drawbacks of these methods constrain their applications, such as poor stability, complex synthesis and time-consuming process. Thus, there is a great need to propose a simple and rapid method to prepare biocompatible, potent and robust antibacterial coatings that can solve the above problems.

Electrostatically assembled coatings have been widely used considering the simplicity of the “synthesis” and the wide range of materials that may be generated with interesting and versatile functions [39]. Moreover, the surface properties of electrostatic assembled coatings can be readily controlled by the choice of charge, ionic strength and different organic solvents [36,40]. In this work, we developed a very effective bactericidal coating consisting of negatively charged poly (γ-glutamic acid) (γ-PGA) and cationic surfactant ethyl lauroyl arginate (ELA) using a novel one-step electrostatic assembly method. The versatility of the coating has been investigated by applying it to inorganic, metal, and polymeric surfaces, respectively. The excellent antibacterial performances of γ-PGA-ELA coatings on the films and the lumen of slender catheters (length up to 2 m, and inner diameter down to 1 mm) under static and dynamic flow conditions were demonstrated by series of experiments in vitro. Good compatibility of the γ-PGA-ELA coatings was proven by CCK-8 cytotoxicity test and hemolysis test. Finally, the histocompatibility and anti-infective properties of the coatings in vivo were studied in a mouse subcutaneous implantation model.

2. Experimental

2.1. Materials

Ethyl lauroyl arginate (ELA, 97 %) was purchased from Aofei Biochemical Co., Ltd. Poly (γ-glutamic acid) (γ-PGA, ~100,000 Da) was provided by Nanjing Bioshineking Biotechnology Co., Ltd. Other chemicals (AR grade) were used as received directly without further purification. The films and medical catheters (0.99/1.25 mm ID/OD) used for the in vitro studies were made of thermoplastic polyurethane (TPU) (BASF, Elastollan® 1190A). The intravenous catheters used for in vivo assay were made by BD Company. Polyethylene (PE) used for melt processing was obtained from Sinopec Maoming Petrochemical Co., Ltd. Cell counting kit-8 (CCK-8) was purchased from Boster Biological Technology Co., Ltd. Gram-negative Escherichia coli (E. coli, ATCC 25922) and Gram-positive Staphylococcus aureus (S. aureus, ATCC 6538) were provided by Nanjing Clinic Biological Technology Co., Ltd. Bacteria culture medium was purchased from Dingguo Biological Technology Co., Ltd. L929 murine fibroblasts cell line was obtained from Shanghai ASTRI Cell Resource Center, Chinese Academy of Sciences. Female BALB/c mice (7-8 weeks old) used for in vivo assay were purchased from laboratory animal center, basic medical college of Jilin university. All animal experiments were conducted in accordance with the guidelines of the Animal Care and Ethics Committee of Jilin University.

2.2. Preparation and characterization of the functionalized γ-PGA compound

The preparation process of γ-PGA-ELA was shown in Scheme 1. γ-PGA and ELA were respectively dissolved in ultrapure water to obtain a 1% (m/v) aqueous solution. Then the γ-PGA aqueous solution was slowly dropped into the ELA aqueous solution under stirring, and white precipitation was generated immediately. As the γ-PGA solution was continuously added, the precipitation began to agglomerate and separate from the water. Subsequently, the precipitate was collected and ultrasonically washed with ultrapure water for three times (10 min for each time). The resulting precipitate was freeze-dried to obtain a white solid product. The successful synthesis of γ-PGA-ELA was proved by 1H nuclear magnetic resonance spectroscopy (1H-NMR) and Fourier transform-infrared spectroscopy (FTIR, BRUKER Vertex 70). 1H NMR spectroscopy was conducted on a Bruker AV 400 MHz spectrometer with Methanol-D4 (CD4O) as a solvent. Chemical shifts (δ) were reported in the units of ppm and referenced to the protonic impurities. FTIR spectra with the range of 400-4000 nm were measured in an ATR mode. Total of 32 scans were accumulated with a resolution of 4 cm-1 for each spectrum. The microstructure of γ-PGA-ELA was analyzed by X-ray scattering. in situ wide angle X-ray scattering (WAXS) experiments were performed using a custom-designed micro-focus X-ray beam of a Xeuss system of Xenocs, France. in situ synchrotron small angle X-ray scattering (SAXS) measurements were performed at beamline 1W2A, BSRF, Beijing, China. Thermal stability of γ-PGA-ELA was measured by thermo gravimetric analysis/differential thermal analysis (TGA/DTA, METTLER TOLEDO) which was performed at a heating rate of 10 °C min-1 from 25 °C to 800 °C under an inert atmosphere of N2.

Scheme 1.

Scheme 1.   Schematic of the preparation process of the functionalized γ-PGA compound.


2.3. Preparation and characterization of the functionalized γ-PGA compound coatings and blends

Before preparing the coatings, the solubility of γ-PGA-ELA was studied in different solvents (Table S1 in Suppoting Information). Ethanol was selected as the solvent to dissolve γ-PGA-ELA because of its low toxicity and easy evaporation. The samples to be coated were previously ultrasonically washed three times with ethanol. The coatings constructions on different samples were realized by soaking them into γ-PGA-ELA ethanol solution for 2 min and naturally drying at ambient temperature. Subsequent water washing process was applied to remove uncoated γ-PGA-ELA. The blends of PE with γ-PGA-ELA or ELA were prepared by thermal processing. Polyethylene was premixed with different mass fractions of γ-PGA-ELA or ELA at room temperature. Then the mixture was fully mixed at 150 °C for 10 min in an internal mixer (XSS-300, Shanghai Kechuang Rubber & Plastic Machinery Equipment Co., Ltd.). The next hot pressing process was performed at 180 °C. The final concentration of γ-PGA-ELA in the films (the thickness was ~ 0.8 mm) was 5%, 10 % (w/w). As a contrast, a blend film of PE with 10 % (w/w) ELA was also prepared with the same method. Then the films were washed with excess amount of sterile water. Crystal violet staining assay was used to visualize the formation of γ-PGA-ELA coatings. Positive-charged crystal violet can combine with negative-charged γ-PGA to give a purple coating. Hence, the formation of γ-PGA-ELA coatings can be judged by simple color change. The wettability of the coating surface was studied by the water contact angle measurement. The water contact angle on the γ-PGA-ELA coated TPU films were evaluated using the sessile drop method with a 2 μL water droplet in a DSA100 goniometer (KRÜSS GMBH, Germany). All samples are at least in triplicate for calculating the average value.

2.4. In vitrobiological test

2.4.1. Antibacterial performances of the functionalized γ-PGA compound coatings and blends

Inhibition zone assay [41]. In order to evaluate the bactericidal properties of γ-PGA-ELA coatings, the inhibition zone assay was adopted to qualitatively study the antibacterial mode of γ-PGA-ELA coatings. The specific operation was listed as follows: an overnight bacterial suspension was inoculated in approximate 50 mL of Luria broth (LB) medium at 37 °C with shaking; the concentration of bacteria in LB was adjusted to give an initial optical density (OD) reading of 0.1 at the wavelength of 600 nm on a microplate reader (TECAN SUNRISE, Swiss), which corresponds to the concentration of 1 × 108 CFU mL-1; then the bacterial suspension with a concentration of 1 × 106 CFU mL-1 was obtained by 100 times dilution; 200 μL of the bacterial suspension (1 × 106 CFU mL-1) was uniformly inoculated on the agar plates; the coated films and the control references with diameters of 0.5 cm were gently placed on the agar plates and cultured with bacteria at 37 °C for 24 h. The antibacterial activity of the coatings was roughly determined by the size of the inhibitory region.

Agar plate colony counting assay. The antibacterial activity of the samples (the as-prepared coatings or blends) was quantitatively evaluated by agar plate colony counting assay according to JIS Z 2801 standard. All samples with the size of 1.5 cm × 1.5 cm were placed in a 12-well plate and 25 μL of bacterial suspension (1 × 106 CFU mL-1) was added at the center region of the films. Then the samples were covered with a pristine PE membrane with the size of 1.0 cm × 1.0 cm. After that, a small amount of H2O was added into the wells around the samples, and placed the 12-well plate in a 37 °C incubator to ensure the appropriate temperature and humidity required for normal growth of bacteria. After cultivated for 24 h, 2 mL of PBS buffer was added to each well directly to make sure the samples were entirely immersed. Then the plate was ultrasonicated for 5 min to release the bacteria adherent on the films. After that, PBS buffer containing bacteria was serially diluted and plated for colony counts. The number of the colony-forming units (CFUs) was counted after incubation at 37 °C for 24 h. Each sample was carried out at least in triplicate. The bactericidal efficiency (%) was calculated according to the reference [41].

Morphology of the attached bacteria. The morphology of bacteria attached to the surfaces was observed under field emitted scanning electron microscopy (SEM, XL 30 FESEM FEG, FEI Company, USA). The samples (1.0 cm × 1.3 cm) were placed in a 24-well plate and 500 μL of bacterial solution (1 × 106 CFU mL-1) was added to each well to ensure the samples were just submersed. After cultivated at 37 °C for 24 h, the samples were gently washed with PBS buffer for three times to remove loosely adherent bacteria. Then the samples were removed to another 24-well plate and fixed in paraformaldehyde (4 wt.%, 1 mL) for 2 h, followed by washing three times with PBS and ultrapure water, respectively. After rinsing thoroughly with ultrapure water, the samples were freeze-dried at -50 °C under vacuum. The morphology of bacteria adherent on the films was observed by SEM.

Viability of the attached bacteria. Confocal laser scanning microscopy (CLSM, LSM 700, Carl Zeiss) was used to calculate the ratio of alive and dead bacterial cells adherent on the samples. After cultivation of the samples with bacteria for 24 h, they were washed three times with PBS buffer and H2O to remove loosely adherent bacteria. Then the samples were removed to a new 24-well plate and stained by 25 μL of a LIVE/DEAD baclight viability kit for 20 min in the dark. The samples were then lyophilized under dark and observed by CLSM (LSM 700, Carl Zeiss). The antibacterial assays above were also repeated for E. coli.

Antibacterial activity of intraluminal catheters under static and dynamic flow conditions. For static flow catheter test [42], all TPU catheter pieces were strictly sterilized by 75 % ethanol for 15 min before coating processes and ultraviolet disinfection before experiments. 100 μL of the prepared 1 × 106 CFU mL-1 LB bacterial solution was injected into each lumen of the catheters. The catheters were incubated at 37 °C for 3 h. Then the bacterial solution in the lumen of the catheter was collected into microfuge tubes. On the one hand, the bacterial solution of 30 μL was diluted and plated for colony counting. On the other hand, the remaining bacterial solution of 40 μL was added to the 2 mL of aseptic LB liquid and inoculated into a 96-well plate. The plate was placed in a shaker at 37 °C for 24 h, and the OD values at 600 nm were measured at 0 h, 4 h, 8 h, 12 h, and 24 h. For static flow catheter test [43], the coated catheters were obtained by pumping the γ-PGA-ELA ethanol solution to the sterile catheters using a peristaltic pump (LEAD FLUID, BT101 L). The coated catheters were incubated with bacterial solution (1 × 106 CFU mL-1) at 37 °C for 3 h. Then the bacteria solution was flowed out, and sterilized LB solution was slowly pumped (0.1 mL min-1) through the catheters at 37 °C for 24 h. Subsequently, the catheters were removed from peristaltic pump, and 1 cm long catheters were cut off from both ends and the middle part of the catheters for quantitative counting analysis and SEM imaging analysis.

2.4.2. Compatibility of the functionalized γ-PGA compound coatings

Hemolysis assay. The hemocompatibility of the coatings were investigated via hemolysis assay as described previously by Yang et al. with minor modifications [44]. Fresh mouse blood was obtained and diluted with saline solution to reach a concentration of approximately 5.0 vol.% of the red blood cells (RBCs). The leaching liquor (300 μL) of the coatings in saline was placed in a 1.5 mL microfuge tube, followed by the addition of an equal volume (300 μL) of 5.0 vol.% RBCs suspension. The mixture was then incubated at 37 °C for 1 h, allowing the hemolysis process to take place. Then non-hemolysed RBCs were separated by centrifugation at 3000 rpm for 5 min. Then the supernatant liquor (100 μL) was transferred to a 96-well plate, and hemoglobin release was measured by UV-absorbance at 540 nm using a microplate reader. Two controls were provided in this assay: untreated RBCs suspension in saline solution was used as the negative control, and the solution containing red blood cells lysed with H2O was used as the positive control. Each assay was performed in 4 replicates, and the data were expressed as means and standard deviations of the 4 replicates. Percentage of hemolysis was calculated as the following formula:

Hemolysis(%)$=\frac{OD_{test at 540 nm}-OD_{negtive at 540 nm}}{OD_{positive at 540}-OD_{negtive at 540 nm}}\times 100$%

Cytotoxicity assay. To test the potential of cytotoxicity induced by the coatings, in vitro CCK-8 assay was conducted according to ISO 10993-5. L929 murine fibroblasts cells were seeded in a 96-well plate at a density of 1 × 104 cells for each well and cultured in Dulbecco's modified eagle medium (DMEM) supplemented with 10 vol.% fetal bovine serum (FBS), and 1 vol.% penicillin-streptomycin solution. The γ-PGA-ELA coated films with 6 cm2 superficial areas were soaked in DMEM supplemented with 10 vol.% fetal bovine serum (FBS) and 1 vol.% penicillin-streptomycin solution for 24 h. Then the cells were exposed to varying concentrations of 100 μL leaching liquor. After cultured for 24 h, 10 μL of CCK-8 solution was added to each well. And the plate was further incubated at 37 °C in 5% CO2 for another 2 h. Then the absorbance at 450 nm was measured with a microplate reader. The positive controls were DMEM supplemented with 10 vol.% fetal bovine serum (FBS) and 1 vol.% penicillin-streptomycin solution without cells, and the negative controls were the uncoated films. The cell viability was calculated by the following formula:

Cell viability(%)=$\frac{\text{O}{{\text{D}}_{\text{test at 450 nm}}}}{\text{O}{{\text{D}}_{\text{test at 540 nm}}}}\times 100%$

2.5. In vivo mice subcutaneous model

All animal experiments were conducted in accordance with the guidelines of the Animal Care and Ethics Committee of Jilin University. The in vivo assays were carried out with reference to experimental process of Yu et al. [45]. As shown in scheme S1 in Supporting Information, the tip of the indwelling needle cannula was cut off by about 0.3 to 0.4 cm, and ultrasonically washed with n-hexane and ethanol for 1 h and 30 min, respectively. Then the samples were immersed in 0.5 % (m/v) γ-PGA-ELA ethanol solution for 10 min to form a coating on the surface. For in vivo antibacterial performance studies, the assembled indwelling needles were soaked in an appropriate amount of S. aureus solution (3 × 108 CFU mL-1) for 4 h. Female mice (n = 15, 7-8 weeks old) were anesthetized by intraperitoneal injection of ethyl carbamate according to the body weight. Then the indwelling needle cannulas incubated with bacteria were implanted into the back subcutaneous tissue of the mouse with the coated catheter on the left and the uncoated catheter on the right. Five days later, the mice were euthanized and the associated peri-implant tissue was surgically removed for the histological analysis. Simply, the muscles of the implant sites were removed and immersion-fixed in 4% paraformaldehyde for at least 24 h prior to paraffin embedding and sectioning for routine hematoxylin and eosin staining (H&E stain kit). The slides were visualized with real-time imaging microscope (OLYMPUS IX71). The implanted catheter fragments were transferred into microfuge tube containing 500 μL sterile PBS and ultrasonicated in water bath for 5 min to ensure the dispersion of bacteria in PBS, then each sample was serially diluted and plated on agar plates for CFU analysis. For biocompatibility experiments, the experimental process was basically the same as the antibacterial experiment in vivo, except the implanted catheters were not incubated in S. aureus suspension.

2.6. Statistical analysis

All data are presented as mean ± standard deviation (SD). Each result is an average of at least three parallel experiments. The statistical significance was assessed by analysis of variance (ANOVA), *(p < 0.05), **(p < 0.01), ***(p < 0.001).

3. Results and discussion

3.1. Preparation and characterization of the functionalized γ-PGA compound

γ-PGA is an anionic biopolymer containing carboxyl group. ELA is a cationic surfactant that has been widely studied as food preservatives. The two substances could combine through electrostatic interactions between the carboxyl group of poly glutamate and the guanidine group of ELA (Scheme 1). The product could automatically precipitate out of the solution, and thus the purification steps can be greatly simplified (Fig. S1 in Supporting Information). The structure of γ-PGA-ELA was confirmed by the 1H NMR spectrum shown in Fig. 1(A). Compared with the spectra of γ-PGA and ELA, the 1H NMR spectrum of γ-PGA-ELA showed all the peaks of the two substances at the corresponding positions. Moreover, the enhanced absorption peaks of methylene (-CH2-) at 2850 cm-1 and 2921 cm-1 of γ-PGA-ELA compared with that of γ-PGA as well as the characteristic absorption peak of the carboxylic acid at 3000-3500 cm-1 and a new characteristic absorption peak at 1640 cm-1 (-C=N) in the FTIR of γ-PGA-ELA (Fig. 1(B)) further confirmed the successful combination of γ-PGA and ELA. In addition, γ-PGA and ELA were compounded in an equimolar manner as calculated by the integral of the -CH2- (δ 1.85-1.94 ppm) of γ-PGA compared with the integral of the -CH3 (δ 0.88-0.91 ppm) of ELA.

Fig. 1.

Fig. 1.   Characterization of the functionalized γ-PGA compound. (A) 1H NMR spectra of ELA, γ-PGA and γ-PGA-ELA. The solvents used were CDCl3, D2O and CD4O, respectively. (B) FTIR spectra of γ-PGA, ELA and γ-PGA-ELA. (C) TGA curves of γ-PGA-ELA and γ-PGA.


The microstructure of γ-PGA-ELA was studied by SAXS and WAXS (Fig. S3(a) and (b) in Supporting Information). In the SAXS (Fig. S3(a) in Supporting Information), a sharp diffraction peak appeared at 2.083 nm-1 of ELA and a higher displacement peak appeared at 1.654 nm-1 of γ-PGA-ELA. In analogy to other similar ionic polymer [46,47], this spacing is associated with the periodical distance (L0) of a biphasic layered structure in which the γ-PGA and the surfactant phases are alternating regularly. The WAXS curve (Fig. S3(b) in Supporting Information) of ELA produced multiple discrete scattering in the 0.3-0.5 nm range characteristic of crystalline organic material, while it appeared a broad peak in the WAXS curve of γ-PGA-ELA. It indicates that ELA has changed from crystalline state to amorphous state after incorporation of γ-PGA.

The thermal stability of modified γ-PGA was evaluated by TGA. As shown in Fig. 1(C), the thermal decomposition temperature of γ-PGA-ELA (250 °C) was higher than that of γ-PGA (232 °C), signifying that the thermal stability of γ-PGA had been enhanced after the incorporation of ELA. The good thermal stability of γ-PGA-ELA holds considerable promise for thermal processing with common thermoplastic materials (e.g., polyethylene, polypropylene, polyurethane.).

3.2. Characterization of the functionalized γ-PGA compound coatings

The solubility of γ-PGA-ELA in different solvents was investigated. As shown in Fig. 2(A), the results showed that γ-PGA-ELA was water-insoluble and could be dissolved in various common organic solvents such as methanol, ethanol, tetrahydrofuran, etc. This property provided a good prerequisite for the preparation of coatings. The formation of γ-PGA-ELA coating was visually judged by crystal violet staining assay. Different kinds of substrates were selected, such as inorganic substrates (silicon wafer and glass sheet), metal substrate (aluminum sheet), and polymer substrates (PET film, TPU film, SEBS film and PP non-woven fabric). From Fig. 2(B), the crystal violet on the uncoated substrate was easily washed away; nevertheless a uniform coating was formed on the coated substrates by the electrostatic interaction between γ-PGA and crystal violet. The results demonstrated the universality of the γ-PGA-ELA coating. Notably, a uniform coating could also be formed in the lumen of a slender catheter that is 2 m long and less than 1 mm in diameter (Fig. S2 in Supporting Information).

Fig. 2.

Fig. 2.   Solubility of the functionalized γ-PGA compound and characterization of the γ-PGA-ELA coating. (A) Digital pictures of the functionalized γ-PGA compound dissolved in different solvents. (B) Schematic illustration of the formation of the γ-PGA-ELA coating and digital pictures of different substrates stained with crystal violet; uncoated substrates were treated with the same process as for coated substrates. (C) Water contact angles of untreated PP and PP coated with γ-PGA, ELA, and γ-PGA-ELA. γ-PGA and ELA are water-soluble, and the corresponding coatings could be washed away easily and leave the hydrophobic PP. (D) FTIR spectra of untreated PP film and γ-PGA-ELA coated PP film.


The results of FTIR further confirmed the conclusion above (Fig. 2(D)). The new characteristic absorption peaks at 3271 cm-1 and 1736 cm-1 that represented the absorption of the amide bond (-NH) and the ester bond (-C=O), as well as the absorption peaks at 1640 cm-1 and 1548 cm-1 that represented the absorption of amide I and amide II appeared in γ-PGA-ELA coated PP film, illustrating the successful construction of the γ-PGA-ELA coating on PP film. The surface wettability results (Fig. 2(C)) suggest that PP has a hydrophobic surface with contact angles of approximately 110°. The contact angles of ELA coated PP film and γ-PGA coated PP film were similar to that of PP film since both ELA and γ-PGA were easily dissolved in water during the washing process. However, the contact angles of γ-PGA-ELA coated films (75° ± 3°) decreased obviously compared with that of PP, indicating the enhanced hydrophilicity of the surfaces after the formation of γ-PGA-ELA coating.

3.3. In vitro biological test

3.3.1. Antibacterial performances of the functionalized γ-PGA compound coatings and blends

Antibacterial activity of the functionalized γ-PGA compound coatings. The minimum inhibitory concentration (MIC) of ELA was measured by a standard broth micro-dilution protocol in 96-well cell culture plates [48]. In Fig. S4 in Supporting Information, the results suggested that the MIC of ELA was in the range of 10-25 μg/mL for both S. aureus and E. coli, indicating excellent antibacterial activity of ELA. Fig. 3(A) shows the inhibition zone induced by γ-PGA-ELA coatings at different concentrations. From the results, the coatings had a visible inhibition zone at concentrations as low as 0.2 % (w/v). When the concentration reached 0.5 % (w/v), the coatings showed considerable antibacterial effects. The results of agar plate colony counting assay reflect the bactericidal activity of the coatings quantitatively. As shown in Fig. 3(B), no bacterial colonies were visible to the naked eye on agar plates of the coated films. Moreover, a large number of bacterial colonies grew on agar plates with uncoated films, which indicated the superiority of the coated films in antibacterial activities. The bactericidal rates of the coated films were more than 99.99 % [41] at the concentration of 0.5 % (w/v).

Fig. 3.

Fig. 3.   Antibacterial performances of the functionalized γ-PGA compound coated films. (A) Inhibition zone assay of γ-PGA-ELA coated films at different concentrations. (B) Agar plate colony counting assay: the photographs represent the growth of bacteria on agar plates after co-cultured with samples for 24 h. (C) Representative SEM images of S. aureus adhered to the surface of the uncoated and coated films at different magnifications. (D) Representative CLSM images of S. aureus attachment to the surface of the uncoated and coated films.


In order to further observe the effect of γ-PGA-ELA on bacterial morphology and elucidate the antibacterial mechanism of γ-PGA-ELA, SEM and CLSM experiments were carried out. As shown in Fig. 3(C), the SEM images showed that the adherent bacteria on the surfaces coated with γ-PGA-ELA were obviously reduced, and dead bacteria exhibited significant distorted or damaged membranes. In contrast, the surface of PP had plenty of live bacteria with intact cell morphology except for a few deaths due to natural metabolism. In combination with the results of the inhibition zone experiment and carefully analysis of related similar works [45,49], we propose the following antibacterial mechanism. Firstly, when γ-PGA-ELA coated surface is in close proximity to the bacteria, the pH at the surface near bacteria was between pH 5 and pH 5.5 [50], therefore, the protonated cationic guanidine group of ELA electrostatically attract with the anionic bacteria cell wall. Secondly, as the ELA are attracted to and aggregated on/in the cell wall, the long alkyl chains of ELA insert into the phospholipid bilayer through hydrophobic interaction. Eventually, γ-PGA-ELA induces the rupture of the bacterial cells. The results of CLSM support the mechanism above. In this stain assay, the SYTO-9 dye can label all bacteria with green fluorescence, while PI dye only penetrates bacteria with membrane-compromised cells, displacing the green fluorescence with red. Therefore, the bacteria that stained green are live cells with intact membranes, while bacteria that stained red are dead cells with damaged membranes. As shown in Fig. 3(D), a large number of live bacteria (green fluorescence) adhered to the uncoated surface and formed biofilms. However, the cells treated with γ-PGA-ELA showed strong red fluorescence, which indicated damaged membranes caused by γ-PGA-ELA. The coatings also showed excellent antibacterial activities against Gram-negative bacteria, which demonstrate a broad-spectrum antibacterial activity of the coatings. As shown in Figs. S5 and S6 in Supporting Information, the coating was able to destroy the membranes of Gram-negative bacteria, showing a bactericidal rate as high as 99.99 %.

Catheter-associated infections (CAIs) are often preceded by pathogen colonization on catheter surfaces and represent one of the most common hospital acquired infections with significant economic consequences and increased patient morbidity [42,51]. Fabrication of antibacterial coatings on complex-shaped medical devices is still an extreme challenge, especially for the catheters, as both inner and outer surfaces may require structuring or functionalization. However, the access to these surfaces is hindered and problematic. Thus, it is difficult to modify these surfaces and cleaning or remove of the biofilm is nearly impossible [42,52,53]. At present, most methods [43,54] (such as chemical grafting, plasma treatment, etc.) can only modify and activate materials with simple shapes such as sheets and short tubes. However, it is very difficult to construct antibacterial coatings on the lumen of slender catheters. Thus, a facile, inexpensive, and universal method to fabricate effective antibacterial coatings on complex-shaped medical devices has important clinical values. Here, the modified γ-PGA can form an antibacterial coating on the inner surface of catheters with an aspect ratio more than 2000:1 (more than 2 m in length and less than 1 mm in inner diameter). Under static flow condition, the amount of planktonic bacteria in solution was tested by the OD600 nm changes of the bacterial solution after co-cultivation with the coatings for 3 h. As shown in Fig. 4(A), The OD values increased rapidly for the uncoated catheters while it remained basically unchanged for the coated catheters, indicating that the coating had effective antibacterial activity against the planktonic bacteria in solution. The amount of adherent bacteria on the lumen of the catheter surfaces was also assessed by the agar plate colony counting assay. As shown in Fig. 4(B), no bacterial colonies were observed on the coated catheters, whereas a large amount of bacteria grew on the uncoated samples after 24 h. The antibacterial properties of the coated catheters under dynamic flow environment were also investigated. According to the calculation results shown in Fig. 4(D), γ-PGA-ELA coated catheters could reduce bacterial growth by a log reduction up to 6 in bacterial counts as opposed to uncoated ones. This phenomenon applied to different parts of the catheters, the both sides and the middle part showed similar antibacterial performance compared with uncoated catheters. The photographs represent the growth of bacteria on agar plates of coated catheters and uncoated catheters from different parts of the catheters after 24 h flow of LB were shown in Fig. 4(C), which further confirmed the superior antibacterial performances of the coatings. From the results of SEM (Fig. 4(E)), a large amount of bacteria adhered to the lumen of the uncoated catheter, and biofilms had formed. However, only a few structurally damaged bacteria adhered to the lumen of the coated catheters. The results indicated that the coatings can effectively prevent the colonization of bacteria in the lumen of the catheters, thereby avoiding the occurrence of catheter-related infections.

Fig. 4.

Fig. 4.   Antibacterial activity of the lumen of γ-PGA-ELA coated catheters compared with that of uncoated catheters. S. aureus was selected for the representative bacteria. The catheters were filled with S. aureus solution (1 × 106 CFU mL-1) and cultured at 37 °C for 3 h. For static flow catheter test: (A) OD600 values of the inoculant diluted with fresh LB medium tested at different time. (B) Plates for bacterial count of the S. aureus bacterial suspension diluted with PBS after incubated at 37 °C for 24 h. For dynamic flow catheter test: (C) Photographs represent the growth of bacteria on agar plates of coated catheters and uncoated catheters from different parts of the catheters after 24 h flow of LB. (D) Agar plate colony counting results of the bacteria adhered to the lumen of catheters from front end, middle and tail end, respectively. (E) SEM images of the bacteria adherent on the lumen of the catheters with a total length of 2 m and an inner diameter of 1 mm at both endings and the middle part of the catheters (the yellow arrows indicate the dead bacteria).


To sum up, the coatings not only had antibacterial properties on a regular flat film but also exhibited good antibacterial activity on complex-shaped materials such as biomedical catheters. Beyond that, the antibacterial stability of the coatings is very important for clinical applications. Hence, the antibacterial stability test was carried out under different conditions that mimic the in vivo environment. The results are shown in Fig. S7 in Supporting Information. Approximately 10 cm long catheters were subjected to 0.9 % NaCl, artificial urine, LB and air conditions for an extended period of time. The catheters were removed from the above environments at different time points, and then dried for the antibacterial studies according to the static flow catheter test. The OD600 nm values demonstrated that the samples treated with 0.9 % NaCl, artificial urine and open air conditions retained potency of inhibiting bacterial growth up to 30 days.

Antibacterial activity of PE blended with γ-PGA-ELA. In this study, the modified γ-PGA as antibacterial additives for PE was investigated. PE blended with γ-PGA-ELA showed good antibacterial activity at concentrations of 5% and 10 % (w/w). As shown in Fig. 5(A), PE with 5% (w/w) γ-PGA-ELA exhibited a considerable antibacterial activity with a log reduction up to 3 in bacterial counts, and PE with 10 % (w/w) γ-PGA-ELA showed a better antibacterial activity with a log reduction up to 5. The antibacterial assays of the films composed of PE and ELA were also conducted. As shown in Fig. 5(B), PE blended with ELA also has a good antibacterial effect. Compared to PE blended with γ-PGA-ELA at the same concentration of effective bactericidal group, the antibacterial effect is slightly inferior. This phenomenon may be that some ELA is washed away in the subsequent cleaning process. Thus, PE with γ-PGA-ELA exhibited a better antibacterial stability compared with PE blended with ELA.

Fig. 5.

Fig. 5.   Antibacterial activity PE blended with γ-PGA-ELA. (A) Comparison of antibacterial activities of PE blended with different proportions of γ-PGA-ELA. The photographs represent the number of bacteria growing on the surface of the corresponding membranes after 24 h incubation. (B) Comparison of antibacterial activities of PE blended with 10 % (w/w) γ-PGA-ELA and ELA. The photographs represent the number of bacteria growing on the surface of the corresponding membranes after 24 h incubation.


3.3.2. Compatibility of the functionalized γ-PGA compound coatings

In the medical field, there is great demand for materials with biocompatibility properties, which is also a crucial factor in treatment processes. The biocompatibility of the γ-PGA-ELA coatings was evaluated by CCK-8 cytotoxicity assay and hemolysis assay. The effect of the concentration of the coatings on the biocompatibility was also explored simultaneously. As shown in Fig. 6(A), the cell viabilities of the coatings were close to negative control at concentration of 0.25 %-1.0 %, which indicated that the toxicity of the coatings to the cells could be neglected within this concentration range. Given the expense of the release amount of ELA gradually increased as the concentration increased, it is reasonable for the cell viability to decrease at high concentrations of 2.5 % and 5.0 %. The results of the hemocompatibility of the samples (Fig. 6(B)) against mouse red blood cells (RBCs) were in agreement with the results of cytotoxicity assay, and the hemolysis rate of the coatings increased gradually as the concentration increased. Nevertheless, the coated films showed good hemocompatibility with a hemolysis rate of all concentrations lower than 5%. The good biocompatibility is a prerequisite for applying these coatings to diverse medical devices. In addition, both γ-PGA and ELA are biodegradable [55,56], and their degradation products are nontoxic and harmless amino acids, which could be absorbed or metabolized by the human body, thus ensuring the safety of the coatings in future applications. Combining the above results with the bacterial test results, the coated samples with a concentration of 0.5 % (m/v) were selected for subsequent in vivo experiments.

Fig. 6.

Fig. 6.   Biocompatibility assays of the γ-PGA-ELA coated films with different concentrations. (A) Results of CCK-8 cytotoxicity assay of coated films and control groups against L929 murine fibroblasts cells. (B) Results of hemolysis assay of coated films and control groups against mouse red blood cells.


3.4. In vivo assays

Histocompatibility is a precondition for the application of implantable devices in vivo. The results of the histocompatibility assay of the coated catheters were shown in Fig. 7. After 5 days of normal feeding, no obvious inflammation was observed on the implanted area or surrounding region (Fig. 7(A)), indicating that the coated catheters did not induce an immune reaction and showed good histocompatibility. Additionally, the staining from an H&E assay confirmed the above results. As shown in Fig. 7(B), no lymphatic infiltration was observed around the tissues of the uncoated and coated catheters. The nucleus was evenly distributed on the edge of the cells and no obvious toxicity and inflammatory reactions were observed, demonstrating the commendable histocompatibility of the coated indwelling catheters.

Fig. 7.

Fig. 7.   Results of the histocompatibility assay of coated catheters compared with uncoated catheters based on a sterile subcutaneous implant model. (A) Representative image of the tissue compatibility in visually (5 days) chosen from 5 different samples. (B) Results of H&E staining assay.


The ability of the coatings to hamper bacterial adhesion and subsequent inflammation formation was studied in a mouse model. As shown in Fig. 8(A), significant inflammation was observed at the incisions or around the nearby tissues for the uncoated catheters compared with that observed for the coated catheters. The inflammatory response was subsequently analyzed via tissue images acquired by microscopy. As shown in Fig. 8(C), the morphology of the implant and peri-implant tissues had no difference from the normal muscle tissues. In contrast, diffuse and severe inflammatory infiltrates were observed in the tissue adjacent to all uncoated catheters. In addition, all implants were retrieved and washed with PBS under ultrasonication to determine the number of viable bacteria on the implants by agar plate colony counting assay (Fig. 8(B)). Notably, there were vast numbers of S. aureus growing on the uncoated implants, but few bacteria were retrieved from the coated implants, anticipating that the coated catheters obviously decreased the proliferation of bacteria on the implanted catheters. These results illustrated that the coated catheters maintained excellent antibacterial activity in vivo.

Fig. 8.

Fig. 8.   Antibacterial activity of the coated catheters compared with unmodified catheters in vivo based on a subcutaneous implant model. (A) Representative image of the inflammatory reaction in visually (5 days), chosen from 5 different catheters. (B) Digital photographs and statistical counting of the colonies bred on the agar plates of the implants after a five-day inoculation in the back of the mouse. (C) Results of H&E staining assay of the subcutaneous tissue at the implants.


4. Conclusion

In summary, a multifunctional and universal γ-PGA-ELA coating was proposed via a one-step electrostatic assembly method. The ease and efficacy of the γ-PGA-ELA complex preparation process make it intriguing. The obtained coatings showed potent antibacterial activity against both Gram-positive and Gram-negative bacteria, which can effectively inhibited the bacteria adhesion and the subsequent colonization or biofilm formation on various surfaces, even in the lumen of slender medical catheters (up to 2 m long and down to 1 mm in diameter). The antibacterial results illustrated a log reduction of above 6 on the lumen of catheters under dynamic flow condition. The favorable biocompatibility and stability of γ-PGA-ELA coatings ensure the safety and antibacterial efficiency of its application in vivo. Moreover, γ-PGA-ELA could serve as antibacterial additives mixed with biomedical polymer materials such as PE. Taken together, the γ-PGA-ELA coatings will provide an advantageous strategy to combat medical device-associated infections.

Acknowledgements

This work is financially supported by the National Natural Science Foundation of China (Nos. 51973221 and 51873213), the Youth Innovation Promotion Association of CAS (No. 2017269), the Major Science and Technology Innovation Project of Shandong Province (No. 2019JZZY011105), and the High-Tech Research & Development Program of CAS-WEGO Group.

Appendix A. Supplementary data

Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.jmst.2020.05.017.

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Black silicon is a synthetic nanomaterial that contains high aspect ratio nanoprotrusions on its surface, produced through a simple reactive-ion etching technique for use in photovoltaic applications. Surfaces with high aspect-ratio nanofeatures are also common in the natural world, for example, the wings of the dragonfly Diplacodes bipunctata. Here we show that the nanoprotrusions on the surfaces of both black silicon and D. bipunctata wings form hierarchical structures through the formation of clusters of adjacent nanoprotrusions. These structures generate a mechanical bactericidal effect, independent of chemical composition. Both surfaces are highly bactericidal against all tested Gram-negative and Gram-positive bacteria, and endospores, and exhibit estimated average killing rates of up to ~450,000 cells min(-1) cm(-2). This represents the first reported physical bactericidal activity of black silicon or indeed for any hydrophilic surface. This biomimetic analogue represents an excellent prospect for the development of a new generation of mechano-responsive, antibacterial nanomaterials.

K. Belfield, X. Chen, E. Smith, W. Ashraf, R. Bayston, Acta Biomater. 90 (2019) 157-168.

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Two major complications of indwelling urinary catheterisation include infection and mineral encrustation of the catheter. Our antimicrobial urinary catheter (AUC) impregnated with rifampicin, triclosan, and sparfloxacin has demonstrated long-term protective activity against major uropathogens. This study aimed to firstly assess the ability of the AUC to resist mineral encrustation in the presence and absence of bacteria. Secondly, it aimed to investigate the AUC's anti-biofilm activity against multi-drug resistant organisms. There was no difference in surface roughness between AUC and control segments. In a static and a perfusion model, phosphate deposition was significantly reduced on AUCs challenged with P. mirabilis. Furthermore, none of the AUCs blocked during the 28day test period, unlike controls. The AUC prevented colonisation by methicillin-resistant Staphylococcus aureus, methicillin-resistant Staphylococcus epidermidis, extended-spectrum beta-lactamase producing E. coli, and carbapenemase-producing E. coli for 12 consecutive weekly challenges. All three drugs impregnated into the catheter continued to exert protective activity throughout 12weeks of constant perfusion. The drugs appear to migrate into the crystalline biofilm to continually protect against bacteria not it direct contact with the catheter surface. In conclusion, the AUC reduces mineral encrustation and may increase time to blockage in the presence of P. mirabilis, and does not predispose to mineral deposition under other conditions. It also offers 12weeks of protection against multi-drug resistant bacteria. STATEMENT OF SIGNIFICANCE: Infection and associated mineral encrustation of urinary catheters are two serious complications of indwelling urinary catheters. Others have attempted to address this through various technologies such as coatings, dips, and surface modifications to prevent infection and/or encrustation. However, all current 'anti-infective' urinary catheter technologies are limited to short-term use. Some patients with spinal injuries, multiple sclerosis, stroke survivors and others use long-term catheters for 4-12weeks at a time with multiple catheterisation possibly throughout the rest of their life. We present a urinary catheter for long-term use that is impregnated with three antimicrobials by a patient-protected process to prevent infection and encrustation for up to 12weeks, the maximum lifetime of a long-term catheter before it is changed.

B. Wang, H. Liu, L. Sun, Y. Jin, X. Ding, L. Li, J. Ji, H. Chen, Biomacromolecules 19 (2018) 85-93.

DOI      URL     PMID      [Cited within: 1]

Bacterial infections and biofilm formation on the surface of implants are important issues that greatly affect biomedical applications and even cause device failure. Construction of high drug loading systems on the surface and control of drug release on-demand is an efficient way to lower the development of resistant bacteria and biofilm formation. In the present study, (montmorillonite/hyaluronic acid-gentamicin)10 ((MMT/HA-GS)10) organic/inorganic hybrid multilayer films were alternately self-assembled on substrates. The loading dosage of GS was as high as 0.85 mg/cm(2), which could be due the high specific surface area of MMT. The obtained multilayer film with high roughness gradually degraded in hyaluronidase (HAS) solutions or a bacterial infection microenvironment, which caused the responsive release of GS. The release of GS showed dual enzyme and bacterial infection responsiveness, which also indicated good drug retention and on-demand self-defense release properties of the multilayer films. Moreover, the GS release responsiveness to E. coli showed higher sensitivity than that to S. aureus. There was only approximately 5 wt % GS release from the film in PBS after 48 h of immersion, and the amount quickly increased to 30 wt % in 10(5) CFU/mL of E. coli. Importantly, the high drug dosage, smart drug release, and film peeling from the surface contributed to the efficient antibacterial properties and long-term biofilm inhibition functions. Both in vitro and in vivo antibacterial tests indicated efficient sterilization function and good mammalian cell and tissue compatibility.

B. Wang, T. Jin, Q. Xu, H. Liu, Z. Ye, H. Chen, Bioconjugate Chem. 27 (2016) 1305-1313.

DOI      URL     [Cited within: 1]

C. Yang, X. Ding, R. Ono, H. Lee, L. Hsu, Y. Tong, J. Hedrick, Y. Yang, Adv. Mater. 26 (2014) 7346-7351.

DOI      URL     [Cited within: 1]

An antibacterial and antifouling surface is obtained by simple one-step immersion of a catheter surface with brush-like polycarbonates containing pendent adhesive dopamine, antifouling polyethylene glycol (PEG), and antibacterial cations. This coating demonstrates excellent antibacterial and antifouling activities against both Gram-positive (S. aureus) and Gram-negative (E. coli) bacteria, proteins, and platelets, good stability under simulated blood-flow conditions, and no toxicity.

C. Faul, M. Antoniett, Adv. Mater. 15 (2003) 673-683.

DOI      URL     [Cited within: 2]

Y. Cao, B. Su, S. Chinnaraj, S. Jana, L. Bowen, S. Charlton, P. Duan, N.S. Jakubovics, J. Chen, Sci. Rep. 8 (2018) 1071.

DOI      URL     PMID      [Cited within: 1]

Titanium-based implants are ubiquitous in the healthcare industries and often suffer from bacterial attachment which results in infections. An innovative method of reducing bacterial growth is to employ nanostructures on implant materials that cause contact-dependent cell death by mechanical rupture of bacterial cell membranes. To achieve this, we synthesized nanostructures with different architectures on titanium surfaces using hydrothermal treatment processes and then examined the growth of Staphylococcus epidermidis on these surfaces. The structure obtained after a two-hour hydrothermal treatment (referred to as spear-type) showed the least bacterial attachment at short times but over a period of 6 days tended to support the formation of thick biofilms. By contrast, the structure obtained after a three-hour hydrothermal treatment (referred to as pocket-type) was found to delay biofilm formation up to 6 days and killed 47% of the initially attached bacteria by penetrating or compressing the bacteria in between the network of intertwined nano-spears. The results point to the efficacy of pocket-type nanostructure in increasing the killing rate of individual bacteria and potentially delaying longer-term biofilm formation.

P.M. Tsimbouri, L. Fisher, N. Holloway, T. Sjostrom, A. H.Nobbs, R.M. Meek, B. Su, M.J. Dalby, Sci. Rep. 6 (2016) 36857.

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Nanotopographical cues on Ti have been shown to elicit different cell responses such as cell differentiation and selective growth. Bone remodelling is a constant process requiring specific cues for optimal bone growth and implant fixation. Moreover, biofilm formation and the resulting infection on surgical implants is a major issue. Our aim is to identify nanopatterns on Ti surfaces that would be optimal for both bone remodelling and for reducing risk of bacterial infection. Primary human osteoblast/osteoclast co-cultures were seeded onto Ti substrates with TiO2 nanowires grown under alkaline conditions at 240 degrees C for different times (2, 2.5 or 3 h). Cell growth and behaviour was assessed by scanning electron microscopy (SEM), immunofluorescence microscopy, histochemistry and quantitative RT-PCR methods. Bacterial colonisation of the nanowire surfaces was also assessed by confocal microscopy and SEM. From the three surfaces tested the 2 h nanowire surface supported osteoblast and to a lesser extent osteoclast growth and differentiation. At the same time bacterial viability was reduced. Hence the 2 h surface provided optimal bone remodeling in vitro conditions while reducing infection risk, making it a favourable candidate for future implant surfaces.

C. Bain, P. Claesson, D. Langevin, R. Meszaros, T. Nylander, C. Stubenrauch, S. Titmuss, R. von Klitzing, Adv.Colloid Interface Sci. 155 (2010) 32-49.

[Cited within: 1]

A. Tummino, J. Toscano, F. Sebastiani, B. Noskov, I. Varga, R. Campbell, Langmuir 34 (2018) 2312-2323.

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We demonstrate the ability to tune the formation of extended structures in films of poly(sodium styrenesulfonate)/dodecyltrimethylammonium bromide at the air/water interface through control over the charge/structure of aggregates as well as the ionic strength of the subphase. Our methodology to prepare loaded polyelectrolyte/surfactant films from self-assembled liquid crystalline aggregates exploits their fast dissociation and Marangoni spreading of material upon contact with an aqueous subphase. This process is proposed as a potential new route to prepare cheap biocompatible films for transfer applications. We show that films spread on water from swollen aggregates of low/negative charge have 1:1 charge binding and can be compressed only to a monolayer, beyond which material is lost to the bulk. For films spread on water from compact aggregates of positive charge, however, extended structures of the two components are created upon spreading or upon compression of the film beyond a monolayer. The application of ellipsometry, Brewster angle microscopy, and neutron reflectometry as well as measurements of surface pressure isotherms allow us to reason that formation of extended structures is activated by aggregates embedded in the film. The situation upon spreading on 0.1 M NaCl is different as there is a high concentration of small ions that stabilize loops of the polyelectrolyte upon film compression, yet extended structures of both components are only transient. Analogy of the controlled formation of extended structures in fluid monolayers is made to reservoir dynamics in lung surfactant. The work opens up the possibility to control such film dynamics in related systems through the rational design of particles in the future.

J. Sjollema, S. Zaat, V. Fontaine, M. Ramstedt, R. Luginbuehl, K. Thevissen, J. Li, H. van der Mei, H. Busscher, Acta Biomater. 70 (2018) 12-24.

DOI      URL     PMID      [Cited within: 3]

Bacterial adhesion and subsequent biofilm formation on biomedical implants and devices are a major cause of their failure. As systemic antibiotic treatment is often ineffective, there is an urgent need for antimicrobial biomaterials and coatings. The term

K. Yu, J. Lo, M. Yan, X. Yang, D. Brooks, R. Hancock, D. Lange, J. Kizhakkedathu, Biomaterials 116 (2017) 69-81.

DOI      URL     PMID      [Cited within: 3]

Catheter-associated urinary tract infections (CAUTIs) represent one of the most common hospital acquired infections with significant economic consequences and increased patient morbidity. CAUTIs often start with pathogen adhesion and colonization on the catheter surface followed by biofilm formation. Current strategies to prevent CAUTIs are insufficiently effective and antimicrobial coatings based on antimicrobial peptides (AMPs) hold promise in curbing CAUTIs. Here we report an effective surface tethering strategy to prepare AMP coatings on polyurethane (PU), a common biomedical plastic used for catheter manufacture, by using an anti-adhesive hydrophilic polymer coating. An optimized surface active AMP, labeled with cysteine at the C-terminus (RRWRIVVIRVRRC), was used. The coated PU surface was characterized using ATR-FTIR, XPS and atomic force microscopy analyses. The tethered peptides on the PU catheter surface displayed broad spectrum antimicrobial activity and showed long term activity in vitro. The surface coating prevented bacterial adhesion by up to 99.9% for both Gram-positive and -negative bacteria, and inhibited planktonic bacterial growth by up to 70%. In vivo, the coating was tested in a mouse urinary catheter infection model; the AMP-coated PU catheter was able to prevent infection with high efficiency by reducing the bacteria adhesion on catheter surface by more than 4 logs (from 1.2 x 10(6) CFU/mL to 5 x 10(1) CFU/mL) compared to the uncoated catheter surface, and inhibit planktonic bacterial growth in the urine by nearly 3 logs (1.1 x 10(7) CFU/mL to 1.47 x 10(4) CFU/mL). The AMP-brush coating also showed good biocompatibility with bladder epithelial cells and fibroblast cells in cell culture. The new coating might find clinical applications in preventing CAUTIs.

F. Geyer, M. D’Acunzi, C. Yang, M. Müller, P. Baumli, A. Kaltbeitzel, V. Mailänder, N. Encinas, D. Vollmer, H. Butt, Adv. Mater. 31 (2019), 1801324.

DOI      URL     [Cited within: 2]

Y. Qiao, C. Yang, D. Coady, Z. Ong, J. Hedrick, Y. Yang, Biomaterials 33 (2012) 1146-1153.

DOI      URL     [Cited within: 1]

The development of biodegradable antimicrobial polymers adds to the toolbox of attractive antimicrobial agents against antibiotic-resistant microbes. To this end, the potential of polycarbonate polymers as such materials were explored. A series of random polycarbonate polymers consisting of monomers MTC-OEt and MTC-CH2CH3Cl were designed and synthesized using metal-free organocatalytic ring-opening polymerization. Random polycarbonate polymers self-assembled in solution but appeared highly dynamic; such behaviors are desirable as ready disassembly of polymers at the microbial membrane facilitates membrane disruption. Their activities against clinically relevant Gram-positive (Staphylococcus aureus) and Gram-negative bacteria (E.coli and Pseudomonas aeruginosa) revealed that the hydrophobic-hydrophilic composition balance in polymers are important to render antimicrobial potency. Scanning electron microscopy (SEM) studies indicated microbial cell surface damage after treatment with polymers, and confocal microscopy studies also showed entry of FITC-dextran dye in Escherichia coli as a result of membrane disruption. On the other hand, the polymers exhibited minimal toxicity against red blood cells in hemolysis tests. Therefore, these random polycarbonate polymers are promising antimicrobial agents against both Gram-positive and Gram-negative bacteria for various biomedical applications. (C) 2011 Elsevier Ltd.

H. Yu, L. Liu, H. Yang, R. Zhou, C. Che, X. Li, C. Li, S. Luan, J. Yin, H. Shi, ACS Appl. Mater. Interfaces 10 (2018) 39257-39267.

DOI      URL     PMID      [Cited within: 2]

Antibacterial coatings have been considered as an effective method for preventing the implant-associated infections caused by the bacterial colonization. In this study, we report a water-insoluble polyelectrolyte-surfactant complex, poly(hexamethylene biguanide) hydrochloride-sodium stearate (PHMB-SS) that can be facilely coated onto the surfaces of biomedical catheter and kill the bacteria by releasing the PHMB and prevent the generation of the biofilm. The PHMB-SS-coated surfaces showed better bactericidal activity toward Staphylococcus aureus and Escherichia coli. The PHMB-SS-coated catheters could not only relatively prevent the bacterial colonization in vitro but also in an implant-associated bacterial infection animal model in vivo. Moreover, no significant cytotoxicity and host response were observed in vitro and in vivo, indicating the high biocompatibility of the coating. The water-insoluble antibacterial coating reported in this work represents a novel approach to build a simple and effective coating for the prevention of device-associated infections.

A. Tolentino, S. León, A. Alla, A. Martínez de Ilarduya, S. Mu˜noz-Guerra, Macromolecules 46 (2013) 1607-1617.

DOI      URL     [Cited within: 1]

M. García-Alvarez, J. Alvarez, A. Alla, A. Martínez de Ilarduya, C. Herranz, S. Muñoz-Guerra, Macromol. Biosci. 5 (2005) 30-38.

URL     PMID      [Cited within: 1]

P. Li, Y. Poon, W. Li, H. Zhu, S. Yeap, Y. Cao, X. Qi, C. Zhou, M. Lamrani, R. Beuerman, E. Kang, Y. Mu, C. Li, M. Chang, S. Jan Leong, M. Chan-Park, Nat. Mater. 10 (2011) 149-156.

DOI      URL     PMID      [Cited within: 1]

Despite advanced sterilization and aseptic techniques, infections associated with medical implants have not been eradicated. Most present coatings cannot simultaneously fulfil the requirements of antibacterial and antifungal activity as well as biocompatibility and reusability. Here, we report an antimicrobial hydrogel based on dimethyldecylammonium chitosan (with high quaternization)-graft-poly(ethylene glycol) methacrylate (DMDC-Q-g-EM) and poly(ethylene glycol) diacrylate, which has excellent antimicrobial efficacy against Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus and Fusarium solani. The proposed mechanism of the antimicrobial activity of the polycationic hydrogel is by attraction of sections of anionic microbial membrane into the internal nanopores of the hydrogel, like an 'anion sponge', leading to microbial membrane disruption and then microbe death. We have also demonstrated a thin uniform adherent coating of the hydrogel by simple ultraviolet immobilization. An animal study shows that DMDC-Q-g-EM hydrogel coating is biocompatible with rabbit conjunctiva and has no toxicity to the epithelial cells or the underlying stroma.

H. Yu, L. Liu, X. Li, R. Zhou, S. Yan, C. Li, S. Luan, J. Yin, H. Shi, Chem. Eng. J. 360 (2019) 1030-1041.

DOI      URL     [Cited within: 1]

Y. Lu, Y. Wu, J. Liang, M.R. Libera, S.A. Sukhishvili, Biomaterials 45 (2015) 64-71.

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We report on negatively charged layer-by-layer (LbL) hydrogel films, which turn hydrophobic and bactericidal in response to bacteria-induced acidification of the medium. Single-component hydrogel thin films, abbreviated as PaAALbLs, consisting of chemically crosslinked poly(2-alkylacrylic acids) (PaAAs) with varying hydrophobicity [polymethacrylic acid (PMAA), poly(2-ethylacrylic acid) (PEAA), poly(2-n-propylacrylic acid) (PPAA) or poly(2-n-butylacrylic acid) (PBAA)]. With increasing polyacid hydrophobicity, the hydrogel films showed a decrease in water uptake and an increase in elastic modulus. Both parameters were strongly dependent on pH. At pH 7.4, hydrogels of higher hydrophobicity were more resistant to colonization by Staphylococcus epidermidis, with the PBAA coating showing almost negligible colonization. As the medium became more acidic due to bacterial proliferation, the more hydrophobic PEAALbL, PPAALbL and PBAALbL hydrogels became dehydrated and killed bacteria upon contact with the surface. The killing efficiency was strongly enhanced by the polymer hydrophobicity. The films remained cytocompatible with human osteoblasts, as indicated by the MTS assay and live/dead staining. Our approach exploits bacteria-responsive properties of the coating itself without the involvement of potentially toxic cationic polymers or the release of antimicrobial agents. These coatings thus demonstrate a novel approach to the antibacterial protection of tissue-contacting biomedical-device surfaces.

G. Gao, K. Yu, J. Kindrachuk, D. Brooks, R. Hancock, J. Kizhakkedathu, Biomacromolecules 12 (2011) 3715-3727.

DOI      URL     [Cited within: 1]

Primary amine containing copolymer, poly-(N,N-dimethylacrylamide-co-N-(3-aminopropyl)- methacrylamide hydrochloride) (poly(DMA-co-APMA)), brushes were synthesized on Ti surface by surface-initiated atom transfer radical polymerization (SI-ATRP), in aqueous conditions. A series of poly(DMA-co-APMA) copolymer brushes on titanium (Ti) surface with different molecular weights, thicknesses, compositions, and graft densities were synthesized by changing the SI-ATRP reaction conditions. Cysteine- functionalized cationic antimicrobial peptide Tet213 (KRWWLWWRRC) was conjugated to the copolymers brushes using a maleimide-thiol addition reaction after intial modification of the grafted chains using 3-maleimidopropionic acid N-hydroxysuccinimide ester. The modified surfaces were charactized by X-ray photoelectron spectroscopy (XPS), water contact angle measurements, attenuated total reflectacnce Fourier transform infrared (ATR-FTIR) spectroscopy, atomic force microscopy (AFM), and ellipsometry analysis. The conjugation of the Tet213 onto brushes strongly depended on graft density of the brushes at different copolymer brush compositions. The peptide density (peptides/nm(2)) on the surface varied with the initial composition of the copolymer brushes. Higher graft density of the brushes generated high peptide density (pepetide/nm(2)) and lower number of peptides/polymer chain and vice versa. The peptide density' and graft density of the chains on surface greatly influenced the antimicrobial activity of peptide grafted polymer brushes against Pseudomonas aeruginosa.

V. Levering, C. Cao, P. Shivapooja, H. Levinson, X. Zhao, G. López, Biomaterials 77 (2016) 77-86.

DOI      URL     PMID      [Cited within: 1]

Biofilm removal from biomaterials is of fundamental importance, and is especially relevant when considering the problematic and deleterious impact of biofilm infections on the inner surfaces of urinary catheters. Catheter-associated urinary tract infections are the most common cause of hospital-acquired infections and there are over 30 million Foley urinary catheters used annually in the USA. In this paper, we present the design and optimization of urinary catheter prototypes capable of on-demand removal of biofilms from the inner luminal surface of catheters. The urinary catheters utilize 4 intra-wall inflation lumens that are pressure-actuated to generate region-selective strains in the elastomeric urine lumen, and thereby remove overlying biofilms. A combination of finite-element modeling and prototype fabrication was used to optimize the catheter design to generate greater than 30% strain in the majority of the luminal surface when subjected to pressure. The catheter prototypes are able to remove greater than 80% of a mixed community biofilm of Proteus mirabilis and Escherichia coli on-demand, and furthermore are able to remove the biofilm repeatedly. Additionally, experiments with the prototypes demonstrate that biofilm debonding can be achieved upon application of both tensile and compressive strains in the inner surface of the catheter. The fouling-release catheter offers the potential for a non-biologic, non-antibiotic method to remove biofilms and thereby for impacting the thus far intractable problem of catheter-associated infections.

Y. Su, T. Feng, W. Feng, Y. Pei, Z. Li, J. Huo, C. Xie, X. Qu, P. Li, W. Huang, Macromol. Rapid Commun. 40 (2019), 1900268.

DOI      URL     [Cited within: 1]

K. Lim, R. Chua, B. Ho, P. Tambyah, K. Hadinoto, S. Leong, Acta Biomater. 15 (2015) 127-138.

DOI      URL     PMID      [Cited within: 1]

Catheter-associated urinary tract infections (CAUTIs) are the most common hospital-acquired infections worldwide, aggravating the problem of antimicrobial resistance and patient morbidity. There is a need for a potent and robust antimicrobial coating for catheters to prevent these infections. An ideal coating agent should possess high antimicrobial efficacy and be easily and economically conjugated to the catheter surface. In this study, we report a simple yet effective immobilization strategy to tether a potent synthetic antimicrobial peptide, CWR11, onto catheter-relevant surfaces. Polydopamine (PD) was deposited as a thin adherent film onto a polydimethylsiloxane (PDMS) surface to facilitate attachment of CWR11 onto the PD-functionalized polymer. Surface characterization of the CWR11-tethered surfaces confirmed the successful immobilization of peptides onto the PD-coated PDMS. The CWR11-immobilized PDMS slides displayed excellent antimicrobial (significant inhibition of 5x10(4) colony-forming units of CAUTI-relevant microbes) and antibiofilm ( approximately 92% enhanced antibacterial adherence) properties. To assess its clinical relevance, the PD-based immobilization platform was translated onto commercial silicone-coated Foley catheters. The CWR11-impregnated catheter displayed potent bactericidal properties against both Gram-positive and Gram-negative bacteria, and retained its antimicrobial functionality for at least 21days, showing negligible cytotoxicity against human erythrocyte and uroepithelial cells. The outcome of this study demonstrates the proof-of-concept potential of a polydopamine-CWR11-functionalized catheter to combat CAUTIs.

B. Romberg, J. Metselaar, T. de Vringer, K. Motonaga, J. Kettenes-van den Bosch, C. Oussoren, G. Storm, W. Hennink, Bioconjugate Chem. 16 (2005) 767-774.

DOI      URL     [Cited within: 1]

D. Asker, J. Weiss, D. McClements, Langmuir 25 (2009) 116-122.

DOI      URL     PMID      [Cited within: 1]

Lauric arginate (LAE), a cationic surfactant, is a highly potent food-grade antimicrobial that is active against a wide range of food pathogens and spoilage organisms. In compositionally complex environments, the antimicrobial activity of cationic LAE is likely to be impacted by its interactions with anionic components. The purpose of this study was to characterize the interactions between cationic LAE and an anionic biopolymer (high methoxyl pectin, HMP) using isothermal titration calorimetry (ITC), microelectrophoresis (ME), and turbidity measurements. ITC and ME measurements indicated that LAE bound to pectin, while turbidity measurements indicated that the complexes formed could be either soluble or insoluble depending on solution composition. In the absence of pectin, the critical micelle concentration (CMC) of LAE determined by ITC at 25 degrees C was 0.21% (w/v). The amount of LAE bound per unit amount of pectin decreased with increasing pectin concentration (from 1.5 to 0.5 g/g for 0.05 to 0.5 wt % pectin) and with increasing temperature (from 1.7 to 1.3 g/g for 15 to 40 degrees C). The binding contribution to the LAE-pectin interaction was exothermic and was attributed to electrostatic attraction between the cationic surfactant and anionic biopolymer. This study demonstrates that lauric arginate can form either soluble or insoluble complexes with anionic biopolymers depending on the composition of the system.

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