UV-Responsive Multilayers with Multiple Functions for Biofilm

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Biological and Medical Applications of Materials and Interfaces

UV-responsive multilayers with multiple functions for biofilm destruction and tissue regeneration Zhang Haolan, Danyu Wang, Xingang Zuo, and Changyou Gao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b04428 • Publication Date (Web): 23 Apr 2019 Downloaded from http://pubs.acs.org on April 23, 2019

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ACS Applied Materials & Interfaces

UV-Responsive Multilayers with Multiple Functions for Biofilm Destruction and Tissue Regeneration Haolan Zhang1#, Danyu Wang1#, Xingang Zuo1, Changyou Gao1, 2* 1

MOE Key Laboratory of Macromolecular Synthesis and Functionalization, Department of

Polymer Science and Engineering, Zhejiang University, Hangzhou 310027, China 2

Dr. Li Dak Sum & Yip Yio Chin Center for Stem Cell and Regenerative Medicine, Zhejiang

University, Zheda Road, Hangzhou 310027 China

* Corresponding author: Changyou Gao, Prof. Ph.D, email: [email protected] #

These authors contributed equally to this study.

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Abstract:

The increasing demands of surgical implantation highlight the significance of anti-infection of medical devices, especially antibiofilm contamination on the surface of implants. The biofilms developed by colonized microbes will largely hinder the adhesion of host cells, leading to the failure in long term applications. In this work, UV-responsive multilayers were fabricated by stepwise assembly of poly(pyrenemethyl acrylate-co-acrylic acid) (P(PA-co-AA)) micelles and chitosan on different types of substrates. Under UV irradiation, the cleavage of pyrene ester bonds in the P(PA-co-AA) molecules resulted in the increase of roughness and hydrophilicity of the multilayers. During this process reactive oxygen species (ROS) were generated in situ within 10 s, which destroyed the biofilms of Staphylococcus aureus (S. aureus), leading to the degradation of bacterial matrix. The antibacterial rate was above 99.999%. The UV-irradiated multilayers allowed the attachment and proliferation of fibroblasts, endothelial cells and smooth muscle cells, benefiting the tissue integration of the implants. When polydimethylsiloxane (PDMS) slices with the multilayers were implanted in vivo and irradiated by UV, the density of bacteria and inflammatory level (judging from the number of neutrophils) decreased significantly. Moreover, formation of neo blood vessels surrounding the implants was observed after implantation for 7 d. These results reveal that the photo-responsive multilayers endow the implants with multifunctions of simultaneous anti-biofilm and tissue integration, shedding a light for applications in surface modification of implants in particular for long term use.

Key words: multilayers, photo-responsive, anti-biofilm, tissue regeneration, ROS

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1. Introduction

The medical devices are increasingly used nowadays in clinics, for example, the replacement surgeries for knees and hips.1, 2 The implants, especially those used for an entire lifetime, are usually required to integrate with surrounding tissues to avoid loosening and development of fibrous tissues.3, 4 One of the key issues for implant integration is cell adhesion onto the implant surface as fast as possible.5 Hence, many strategies have been developed to accelerate cell adhesion and tissue integration, such as introduction of surface roughness and topology, and loading of peptides or cytokines, etc.6-8 It is known that the epithelial or mucosal barriers are invaded during implantation, and thus the implants are particularly vulnerable for microbial colonization, leading to a high infection risk.9 Indeed, the infection of implant devices accounts for approximately 45% of all nosocomial infections.10 Although the infection can be remedied by the sustained administration of antibiotics or surgical debridement, the outcome is limited once the adhered bacteria develop into biofilms. The biofilms act as protective barriers against immunity and traditional antibiotics. In biofilms, microbes are encased by a polymeric matrix composing of polysaccharide-cementing substances, DNAs and proteins.2,

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The possible reasons for drug resistance of biofilms include incomplete penetration of anti-biotic through biofilm matrix and intercellular communication, which is referred to as “quorum sensing”.12, 13 Under the worst circumstance of biofilm affection, the implants must be removed. On one hand, the infection of bacteria hinders the integration between normal tissues and implants, and evokes severe immune responses.14 On the other hand, the quick adhesion of host cells not only accelerates the tissue-material integration, but also helps increase resistance of bacterial colony, and thus can largely avoid infection-caused loosening.15 It is thus critical to consider both infection 3



prevention and tissue integration to achieve long term application of implants in vivo. Besides antibiotics, many anti-microbial coatings have emerged for protecting biomaterials from biofilm infection and promoting tissue regeneration. One of the major strategies is to prevent the formation of biofilms on biomaterials. The antifouling molecules, such as poly(ethylene glycol) (PEG) and zwitterionic polymers are widely used to resist microbial attachment.16 However, the adhesion of cells is also blocked to a greatest extent, which is disadvantageous to the integration of implants. To solve this problem, mammal cells-specific adhesive molecules can be incorporated onto the antifouling surfaces17,

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with bacterial responsive property in some cases.19 Other molecules

specifically targeting microbes such as cationic polymers20 and antimicrobial peptides21 have also been developed, showing excellent biocompatibility as well. Because the infection and integration of implants with tissues take place mainly on the surface, it would be very desirable to modify the surface properties of implants without influence on their bulk properties, realizing the simultaneous anti-biofilms and tissue growth. In this study, a multilayer coating is designed for destruction of mature biofilms through the generation of ROS under UV-irradiation, which then promotes cell adhesion and tissue regeneration (Figure 1). Poly(pyrenemethyl acrylate-co-acrylic acid) (P(PA-co-AA)) is assembled with chitosan (CS) to form multilayers on a substrate surface. In the scenario of biofilm infection, the pyrene ester bonds are cleaved under the irradiation of 365 nm UV light, generating massive ROS which kills bacteria and degrades the matrix of biofilms. Moreover, the transferred surface is suitable for adhesion and proliferation of mammal cells. The performance of multilayers is highlighted by the thorough elimination of mature biofilms, which are hard to be removed in clinics.

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7Sea Biotech Co. Ltd. Other chemicals were of analytical grade and used as received. S. aureus strain, human endothelial cells (ECs), human smooth muscle cells (SMCs) and human pulmonary fibroblasts (FIBs) were obtained from American Type Culture Collection (ATCC). Sprague-Dawley (SD) male rats were provided from Zhejiang Academy of Medical Sciences. The water used in all experiments was purified by a Millipore system, and had a resistivity higher than 18.2

L1"#1

2.2 Fabrication of multilayer coating

The synthesis of P(PA-co-AA) and preparation of P(PA-co-AA) micelles were performed according to our previous work.22 Photoresponsive micelles-containing multilayers were fabricated by layer-by-layer (LbL) assembly of P(PA-co-AA) micelles and chitosan on different substrates. The substrates were cleaned by different methods. The glass slides (0.9 cm×0.9 cm) and quartz slides were soaked in a piranha solution (70% H2SO4+30% H2O2) overnight, washed with plenty of water, and finally dried in a 60 oC oven. The PDMS round slices (diameter, 1 cm) were soaked in ethanol under ultrasonication for 15 min and dried at room temperature. They were then etched with air plasma under a pressure of 2.1-2.3 Pa for 10 min to enable the surface with negative charge. To prepare the multilayers, all these pre-treated substrates were incubated in 2 mg/mL chitosan/1% acetic acid solution for 5 min, washed with water for 3 times, and dried with a nitrogen flow. They were then dipped in 0.5 mg/mL P(PA-co-AA) micelles/0.1M NaCl solution (pH was adjusted to about 5.1) for 5 min, washed with water for 3 times, and dried with a nitrogen stream. The multilayers were obtained by repeating these steps alternately. For all the biological experiments, 6 bilayers of P(PA-co-AA)/CS were used.

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2.3 Physicochemical characterizations

The assembly process was monitored by the absorbance growth on a quartz substrate with a UV-Vis spectrometer (UV-2550, Shimadzu). The assembly of multilayers on PDMS slices was characterized with an attenuated total reflection-infrared (ATR-IR) spectrometer (NICOLET 6700, Thermo). For all the UV irradiation process, substrates were placed in 500 O water and irradiated by 365 nm light (LAMP Technology) with a power of 50 mW/cm2. The irradiation time was set as 5 min except for ROS detection. The surface roughness of the multilayers before and after UV irradiation was measured with atomic force microscopy (AFM) (Nanoscope V Multimode Atomic Force Microscope, Bruker). The wettability of the pristine and UV-irradiated multilayers was analyzed by a static water contact angle measurement system (DSA 100, Krüss) using a sessile-drop method. The volume of each droplet was 2 O 1 Each measurement was repeated for 3 times. The generation of ROS was measured according to a method reported previously.23 p-CBA (10 O ? and FFA (0.85 mM) were used to detect •OH and 1O2, respectively. The multilayers-coated glass slides were placed in 500 O aqueous solution containing the indicators, followed by UV exposure for different time period (0-3 s, 3-6 s, 6-10 s, 10-20 s, 20-50 s, 50-100 s, 100-150 s, 150-200 s). After each exposure the solution was refreshed. The residual p-CBA and FFA in solution were analyzed with high-performance liquid chromatography (HPLC, HPLC-515, Waters) equipped with an Agilent Zorbax RX-C 18 column and a diode-array UV detector (230 nm for p-CBA and 238 nm for FFA). p-CBA and FFA were eluted using a mixture of methanol (HPLC grade, Sigma-Aldrich) and water at 55:45 (v/v). Both •OH and 1O2 react with their corresponding indicators in a molar ratio of 1:1. In the control experiment, the glass sides with multilayers were substituted with bare ones of the same size, while the process was kept same as the experimental groups. The average relative release rate (v) was 7



calculated by the following equation:

where v is the average relative release rate (s-1) of each type of ROS, C0 is the initial concentration of indicators (mol/L), Ct is the concentration of the indicators after certain UV exposure time period (mol/L), and t is the length of irradiation time (s).

2.4 Groups and procedures for antibacterial studies

For brevity, different groups and related procedures are described below: (i) Multilayers: multilayers-coated substrates covered with biofilms; (ii) Multilayers-UV: multilayers-coated substrates covered with biofilms, followed by exposure to UV light; (iii) Glass: pure glass slides covered with biofilms; (iv) Glass-UV, pure glass slides covered with biofilms, followed by exposure to UV light; (v) Penicillin: pure glass slides covered with biofilms, followed by immersion in 1 mL penicillin solution (104 units/mL) for 1 h; (vi) Control: tissue culture polystyrene (TCPS) without biofilm for in vitro tests, or PDMS slices without biofilm for in vivo tests. The UV light exposure process was as follows: the substrates were placed in 500 O

phosphate buffered saline (PBS) and irradiated by 365 nm light

source (LAMP Technology) for 5 min with a power of 50 mW/cm2. The substrates of all groups were rinsed with sterilized PBS after formation of biofilms and UV irradiation, respectively.

2.5 Antibacterial experiments in vitro

Bacteria culture and biofilm harvesting. S. aureus were expanded with LB medium and suspended in PBS under 4 °C before use. For the biofilm formation, different substrates were immersed in 1 mL LB medium dispersed with S. aureus in a concentration of 107 colony forming unit (CFU)/mL. For robust

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biofilm formation in short incubation time, additional glucose (2 wt %) was added into the LB medium.24 After incubated in an oven at 37 °C for 24 h, biofilms on the substrates were harvested. Different procedures for each group were conducted according to Section 2.4. Bacterial viability measured by fluorescent staining. The viability of bacteria on the substrates was characterized with a LIVE/DEAD BacLight Bacterial Viability Kit. Live and dead bacteria were stained with green and red fluorescent colors, respectively. The images of stained biofilms were captured by a confocal laser scanning microscope (CLSM, LSM-510, Zeiss), and 3D reconstruction images were processed with ZEN 2010 software. Biofilm morphology characterized by SEM. The morphology of the biofilms was examined by scanning electron microscopy (SEM, S-4800, Hitachi). Before measurement, the biofilms were fixed by 4% paraformaldehyde/PBS for 1 h, and then were dehydrated with ethanol solutions with gradient concentrations (30%, 50%, 70%, 80%, 90%, 100%, ethanol: water, v: v) successively, and finally dried in a 37 °C oven overnight. Bacterial viability quantified by colony counting. The live bacteria remained on substrates were quantified according to the standard plate counting assay. In brief, each sample was placed in a centrifuge tube containing 1 mL sterile PBS, where bacteria were thoroughly dispersed from biofilms to PBS by vortex. The suspensions of bacteria were serially diluted with sterile PBS. 100 O diluted samples were spread on growth agar plates. The bacterial colonies on agar plates were counted after incubation at 37 °C for 12 h. Each group had 3 replicate samples. The antibacterial rate was calculated according to the following equation25:

where CFU experimental group and CFU control group represent the CFU of the experimental and control groups,

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respectively.

2.6 Cell experiments

Cell culture. ECs, SMCs and FIBs were cultured in high glucose Dulbecco's Modified Eagle Medium (DMEM) and maintained at 37 °C in a humidified incubator (Forma Series II, Thermo Fisher Scientific Inc., USA) containing 5% (v/v) CO2. The culture medium was changed every 2-3 d. Cell adhesion assay. ECs, SMCs and FIBs were labeled with 1 O K# calcein AM (Yeasen Co. Ltd.) for 30 min. The samples in Control, Multilayer-UV, Multilayers, Glass, Glass-UV and Penicillin groups were prepared and processed according to Section 2.4, respectively. All the substrates were then placed in sterile 24-well plates. Into each well 1 mL of culture medium (containing 85% antibiotic-free DMEM and 15% glucose-containing LB medium) with 3×104 cells was added. After incubation for 24 h, the unattached cells were washed off with PBS. Cells adhered on the surface were recorded under a fluorescence microscope (IX81, Olympus). At least 10 images were randomly recorded, and 3 parallel samples were determined. The number and average adhesion area of cells remained on the substrates were calculated with the Image Pro Plus software. Cell viability. The samples in Control, Multilayer-UV, Multilayers, Glass, Glass-UV and Penicillin groups were prepared and processed according to Section 2.4, respectively. They were then placed in sterile 24-well plates. Into each well, 1 mL of culture medium (containing 85% antibiotic-free DMEM and 15% glucose-containing LB medium) with 1×104 cells was added. After incubation for 1, 3 and 5d, the cells were detached with trypsinization. The cells and bacteria were separated by centrifugation-resuspension in DMEM with 800 rpm for 3 times. Finally, the cells in each sample were resuspended by 1 mL DMEM containing 50 O CCK-8, and incubated in 24-well microplate for

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another 4 h. The absorbance at 450 nm was measured by a microplate reader (Infinite M200 Pro, Tecan). 3 parallel samples were conducted.

2.7 In vivo test

Implantation process. Subcutaneous implantation of the PDMS slices into male SD rats (200 g) was carried out to assess the anti-bacteria performance in vivo. The experiments were approved by the Institutional Animal Care and Use Committee of Zhejiang University. The PDMS slices were divided into three groups: (i) Control, pure PDMS slices; (ii) Multilayers, multilayers-coated PDMS slices covered with S. aureus biofilms; (iii) Multilayer-UV, multilayers-coated PDMS slices covered with S. aureus biofilms, with UV irradiation 24 h post implantation. The samples of three groups were randomly implanted in the upper, middle or bottom part of the back of each rat, and 5 parallel samples were set for each group. 24 h after implantation, the incisions of Multilayers-UV group were opened, and the implants were irradiated by UV (50 mW/cm2, 5 min), followed by suture. Meanwhile, the incisions of control and Multilayers groups were opened also, but were then sutured without UV irradiation. After surgery all rats were fed for another 7 d. Gross view. After the rats were sacrificed, the gross view of implants was recorded by a digital camera. Hematoxylin and eosin (H&E) staining. The tissues around the PDMS slices were harvested and fixed in 4% formaldehyde solution for 1-3 d. The fixed tissues were sectioned and stained with a standard H&E method. The images of sections were taken by a microscope (IX81, Olympus). Bacteria quantification. The live bacteria remained in tissues around PDMS implants were quantified according to the standard plate counting assay. Tissues in each group was harvested and weighed, and

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then homogenized and thoroughly dispersed in sterile PBS. The suspensions were then serially diluted with PBS, and 100 O diluted samples were spread on the growth agar plates. The bacterial colonies on agar plates were counted after incubation at 37 °C for 12 h. Each group had 5 replicate samples.

2.8 Statistical analysis

For all statistic data, 3 or more independent experiments were carried out. Statistical analysis was performed based on one-way analysis of variance (ANOVA) with a Tukey post hoc method. Significance values were set at * p

UV-Responsive Multilayers with Multiple Functions for Biofilm

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