Temperature-Responsive Hierarchical Polymer Brushes Switching

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Temperature-Responsive Hierarchical Polymer Brushes Switching from Bactericidal to Cell Repellency Xianghong Wang, Shunjie Yan, Lingjie Song, Hengchong Shi, Huawei Yang, Shifang Luan, Yubin Huang, Jinghua Yin, Ather F Khan, and Jie Zhao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b09968 • Publication Date (Web): 07 Nov 2017 Downloaded from http://pubs.acs.org on November 10, 2017

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ACS Applied Materials & Interfaces

Temperature-Responsive Hierarchical Polymer Brushes Switching from Bactericidal to Cell Repellency Xianghong Wang,†,§ Shunjie Yan,† Lingjie Song,*,† Hengchong Shi,† Huawei Yang,† Shifang Luan,† Yubin Huang,† Jinghua Yin,† Ather Farooq Khan,⊥ Jie Zhao*,‡ †

State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of

Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, People’s Republic of China ‡

Key Laboratory of Bionic Engineering, Ministry of Education, Jilin University,

Changchun 130022, People's Republic of China §

University of Chinese Academy of Sciences, Beijing 100049, People’s Republic of

China ⊥

Interdisciplinary Research Centre in Biomedical Materials, COMSATS Institute of

Information Technology, Defence Road, Off. Raiwind Road, Lahore 54000, Pakistan

∗

Corresponding authors.

Email address: [email protected] (L. Song) and [email protected] (J. Zhao). 1

ACS Paragon Plus Environment


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KEYWORDS: surface-initiated photoiniferter-mediated polymerization (SI-PIMP), hierarchical structure, temperature-responsive, bactericidal, bacterial repellency

2


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ABSTRACT: Unlike conventional poly(N-isopropylacrylamide) (PNIPAM)-based surfaces switching from bactericidal activity to bacterial repellency upon decreasing temperature, we developed a hierarchical polymer architecture, which could maintain bactericidal activities at room temperature while present bacterial repellency at physiological temperature. In this architecture, a thermo-responsive bactericidal upper layer consisting of PNIPAM-based copolymer and vancomycin (Van) moieties was built on an antifouling poly(sulfobetaine methacrylate) (PSBMA) bottom layer via sequential surface-initiated photoiniferter-mediated polymerization (SI-PIMP). At room temperature below the lower critical solution temperature (LCST), the PNIPAM-based upper layer was stretchable, facilitating contact-killing of bacteria by Van. At physiological temperature (above the LCST), PNIPAM-based layer collapsed, thus leading to the burial of Van and exposure of bottom PSBMA brushes, finally displaying notable performances in bacterial inhibition, dead bacteria detachment and biocompatibility, simultaneously. Our strategy provides a novel pathway in the rational design of temperature-sensitive switchable surfaces, which shows great advantages in the real-world infection-resistant applications.

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1. INTRODUCTION Proliferation of pathogenic bacteria and biofilm formation on implantable material surfaces are major causes of device-associated infections, which can lead to severe failures of medical treatment with soaring economic sequelae.1-2 Despite advanced sterilization and aseptic techniques, infections associated with the contamination of medical implants during storage have never been completely eradicated.3 Hence, inhibition of bacterial adhesion, commonly referred as biopassive (bio-resistant) method, is generally regarded as a critical step to prevent the following bacterial infections and biofilm formation.4-7 Although most of bacteria can be repelled by the biopassive surface, some bacteria still have a chance to attach onto the surface to form a stubborn biofilm.8 On the other hand, active biocidal surfaces also demonstrate great potential in preventing biofilm formation via covalent conjugation or physical adsorption/entrapment of bactericides (antibiotics, antimicrobial peptides, cationic polymers, silver ion, etc.).9-13 Unfortunately, continuous contamination by dead bacteria or/and debris can largely reduce the biocidal activities by blocking antimicrobial groups.14 Therefore, synergistic strategies integrating antifouling and bactericidal functionalities have been developed to significantly enhance antibacterial performances and biofilm inhibition.15-21 Recently, one promising antibacterial surface switching from biocidal activity to antifouling performance has been developed via a “kill-and-release” strategy.22-24 Such surface can kill bacteria once they attached, and then switch to antifouling surface upon appropriate stimulus to release dead bacteria and debris on-demand, thus 4


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maintaining long-term antibacterial activity and effective biocompatibility, which shows great attractive for devices that to be ultimately introduced in living body.25 Jiang et al. firstly proposed to build an antimicrobial surface based on a cationic ester, which was able to kill the attached bacteria and release them upon the additional hydrolysis of ester groups.26 In addition, temperature-responsive poly(N-isopropylacrylamide) (PNIPAM) polymer is widely used to control the adhesion behavior of cells, showing great potential in fabricating switchable antibacterial surface.27-34 The tunable property is mainly originated from its reversible and sharp hydrophilic-hydrophobic transition at lower critical solution temperature (LCST) of around 32 °C in the physiological range.35-38 For instance, Gabriel P. López et al. developed a nanopatterned smart surface with the capabilities of attracting, killing and releasing bacteria by integration of PNIPAM brush and biocidal quaternary ammonium salt (QAS).39 Above the LCST, the collapsed PNIPAM region facilitated the exposure of QAS moieties for bacterial killing, while the hydrated and swollen PNIPAM chains promoted the release of bacteria at a reduced temperature below the LCST. Like most PNIPAM-based switchable surfaces reported to date, the transition from bactericidal to bacterial repellency was realized by decreasing temperature, which was contrary to the requirements of medical devices that need bactericidal activities at room temperature but bio-repellency under physiological conditions.33, 39-41 Herein, we presented a temperature-responsive hierarchical surface that could switch from bactericidal properties at room temperature to bacterial repellency and 5



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biocompatible performances at physiological temperature. Specifically, an antifouling zwitterionic

PSBMA

bottom

layer

and

a

thermo-responsive

poly(N-isopropylacrylamide-co-2-carboxyethyl acrylate) P(NIPAM-co-CEA) upper layer were sequentially grafted from silicon wafer via twice SI-PIMP technique to form the hierarchical architecture.42-46 As a powerful glycopeptide antibiotic, Van could selectively kill most Gram-positive bacteria by interfering with the biosynthesis of bacterial cell walls.47, 48 Therefore, Van groups were covalently bonded onto the P(NIPAM-co-CEA) brushes to exert selective antibacterial activity. The surface properties could be readily modulated by changing temperature (Scheme 1). At room temperature below LCST, the PNIPAM-based chains were hydrophilic and stretchable to facilitate the exposure of Van and contact-killing of bacteria. When the temperature was above the LCST, dehydrated PNIPAM-based upper chains would collapse and bury the Van, while the hydrophilic bottom layer of PSBMA tended to migrate towards outmost surface, imparting the surface with biocompatibility.49 Moreover, the conformational change and reorganization of hydrophilic/hydrophobic balance at the outmost surface promoted the release of dead cells and debris, suppression of planktonic bacteria attachment, as well as weakened the cytotoxicity of PNIPAM. The biological performances of the temperature-triggered switchable surface were systematically investigated by a series of tests, e.g., bactericidal efficacy, bacterial adhesion, hemolysis as well as cytotoxicity.

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Scheme 1. Schematic Diagram of the Temperature-Responsive Hierarchical Antibacterial Surface.

2. EXPERIMENTAL SECTION 2.1. Materials Silicon wafers (STM Inc., Singapore) were used as pristine substrates. N,N-diethyldithiocarbamate ethyl]dimethyl-(3-sulfopropyl)

sodium, ammonium

[2-(Methacryloyloxy)

hydroxide

inner

salt

(SBMA),

N-isopropylacrylamide (NIPAM), 2-carboxyethyl acrylate (CEA), vancomycin (Van) 2-morpholinoethanesulfonic

acid

(MES),

N-hydroxysuccinimide

(NHS)

and

1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) were purchased from Sigma-Aldrich

(USA).

p-(chloromethyl)

phenyltrimethoxysilane

and

3-[4,5-Dimethyl-thiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) were obtained from Alfa Aesar Co. (USA). Dulbecco’s modified Eagle’s medium (DMEM) and 0.25 wt. % trypsin were purchased from Beijing Solarbio Science & Technology (China). 7



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Sterile filtered fetal bovine serum (FBS) was supplied by Beijing Yuanhengjinma Biotechnology (China). L929 murine fibroblasts cell line was procured from American Type Culture Collection (ATCC, USA). Gram-positive Staphylococcus

aureus (S. aureus; ATCC 6538), Gram-negative Escherichia coli (E. coli; ATCC 25922), luria-bertani (LB) broth, trypticase soy broth (TSB), alamarBlue and phosphate buffered solution (PBS; 0.1 mol L-1, pH = 7.4) were obtained from Dingguo Biotechnology (China). LIVE/DEAD Backlight Bacterial Viability Kit L7012 was purchased from Molecular Probe Inc. (USA). Unless otherwise specified, all reagents were used as received without further purification. 2.2. Immobilization of Photoiniferter on Silicon Wafers Silicon wafers (∼ 0.5 cm × 0.5 cm) were immersed into a freshly prepared “piranha solution” (H2O2/concentrated H2SO4, 30:70, v/v) at 110 °C for 30 min, rinsed with copious amount of distilled water, and dried by nitrogen flow. Then the wafers were placed into 30 mL anhydrous toluene solution containing p-(chloromethyl) phenyltrimethoxysilane (2 vol. %) for 24 h to generate a self-assembled benzyl chloride monolayer. After being rinsed with toluene, the treated wafers were soaked in 20 mL ethanol solution containing N, N-diethyldithiocarbamate sodium (10 %, w/v) at room temperature for 2 days to obtain photoiniferter moiety-modified silicon samples (denoted as Si-SBDC). 2.3. Preparation of One-Layer and Hierarchical Surfaces To conduct the photopolymerization, the monomer SBMA aqueous solution (10 %, w/v) was firstly degassed with nitrogen flow, then added onto Si-SBDC samples, and 8


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covered with a piece of quartz plate. Subsequently, the “sandwich system” was subjected to UV exposure for 8 min (400 W high-pressure mercury lamp, ~380 nm) to get

the

single-layer

surfaces

(Si-g-PSBMA).

Similarly,

the

single-layer

P(NIPAM-co-CEA)-based copolymer brush surfaces were fabricated by using the NIPAM/CEA comonomer solution (10 wt. % in aqueous solution) under 4 min UV exposure. For the hierarchical brush surfaces, photopolymerization was reinitiated by adding the NIPAM/CEA comonomer solution (10 wt. % in aqueous solution) onto the single-layer Si-g-PSBMA surfaces, followed by 4 min UV exposure. The resultant samples, denoted as Si-g-PSBMA-b-P(NIPAM-co-CEA), were rinsed with water and dried for subsequent analysis. 2.4. Covalent Immobilization of Van to CEA-Containing Samples The details of covalent immobilization of Van onto CEA-containing samples (Si-g-P(NIPAM-co-CEA), Si-g-PSBMA-b-P(NIPAM-co-CEA)) were listed as follows: CEA-containing samples were activated with MES solution containing 0.4 M EDC and 0.1 M NHS at 4 °C for 1 h, rinsed with MES solution and dried by nitrogen flow. Then, the activated samples were placed into the Van aqueous solution (1 mg mL-1) at 25 °C for 3 h to get the resultant samples of Si-g-P(NIPAM-co-Van) and Si-g-PSBMA-b-P(NIPAM-co-Van), respectively. 2.5. Surface Characterization Water contact angles (WCAs) were characterized by a sessile-drop method with a contact-angle goniometer drop-shape analysis (KRÜSS GMBH, Germany) at room temperature (25 °C) and physiological temperature (37 °C). Prior to WCA test at 9



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37 °C, samples were preheated by an electric heater and stabilized. WCA was recorded after 2 µL water droplet added. Moreover, advancing (θa) and receding (θr) water contact angles were also collected and analyzed automatically while water was added and withdrawn from the drop respectively with an automatic dispenser. At least five measurements were performed for calculating the average value. The surface composition was determined by X-ray photoelectron spectroscopy at room temperature (XPS, VG Scientific ESCA MK II Thermo Avantage V3.20 analyzer equipped with an Al Kα anode mono-X-ray source (hν = 1486.6 eV)). The spectra were scanned over a range of 0-1200 eV, and high-resolution C1s spectra were collected. The atomic concentrations of the elements were calculated by the peak-area ratios. 2.6. Antibacterial Activities at Different Temperatures In this test, S. aureus and E. coli were used as representative Gram-positive and Gram-negative bacterial strains. At first, S. aureus or E. coli was inoculated onto separated agar plates, incubated overnight at 37 °C. A single colony of each bacterium from the agar plate was used to inoculate 70 mL TSB at 37 °C for 12 h. The bacteria containing growth broth were then centrifuged at 3000 rpm for 10 min to remove the supernatant. Bacterial cell concentration was calculated by testing the absorbance of cell dispersions at 540 nm using a microplate reader (TECAN SUNRISE, Swiss). An optical density of 1.0 at 540 nm was equivalent to ~109 cells mL-1. For antibacterial assay, the sterilized samples were pre-equilibrated with PBS at 25 °C or 37 °C. The bacterial cells were diluted with PBS to obtain a concentration of 10


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107 cells mL-1. The pre-equilibrated samples were then incubated with 1 mL of diluted bacterial suspensions at different incubation temperature (25 °C or 37 °C) for 6 h and briefly washed with PBS at the same temperature to remove the loosely attached bacteria. The antibacterial capabilities were investigated by both confocal laser scanning microscopy (CLSM, LSM 700, Carl Zeiss) and field-emitted scanning electron microscopy (SEM, XL 30 FESEM FEG, FEI Co.). For CLSM examination, the bacteria on the samples were stained by LIVE/DEAD BacLight Viability Kit for 15 min in the dark, followed by washing with sterile water and vacuum freeze dehydration. The CLSM images were analyzed using ImageJ software. The killing efficiency was determined by dividing the area of dead bacteria to the total area of bacteria. For SEM examination, the adherent bacteria on the samples were fixed with 4 vol. % paraformaldehyde for 4 h. After dehydrating by sequential immersion steps in a series of ethanol aqueous solution (10, 30, 50, 70, 90, 100 vol. %, each for 15 min), the morphology and number of adherent bacteria were evaluated by SEM. 2.7. Bacteria Releasing Assay Two groups of samples were pre-equilibrated at 25 °C, followed by 6 h incubation in bacterial suspensions (1 mL, 107 cells mL-1) at 25 °C. After being gently washed with PBS at 25 °C, one batch of samples were transferred into 5 mL PBS in a shaker at 37 °C for 1 h with a speed of 120 rpm and then gently rinsed with PBS at 37 °C. As reference, another batch of samples were incubated and rinsed with PBS at 25 °C with the similar procedure. The amount and viability of the bacteria remained on the 11



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samples were evaluated by both CLSM and SEM. 2.8. Platelets and Erythrocytes Adhesion Prior to test, all samples were equilibrated with PBS overnight. Fresh blood was extracted from a healthy rabbit in accordance with the guidelines issued by the ethical committee of the Chinese Academy of Sciences. The blood was centrifuged at 1000 rpm for 15 min to obtain the platelet-rich plasma (PRP) and erythrocyte concentrates. The erythrocytes were resuspended in PBS to obtain erythrocytes suspension at 20 % hematocrit (v/v). Subsequently, 30 µL of fresh PRP or erythrocytes suspension was added onto the sample and incubated for 60 min at 37 °C. After being rinsed with PBS, the adhered platelets or erythrocytes were fixed by paraformaldehyde (4 vol. %, 4 h). The fixed samples were washed with PBS, dehydrated by sequential immersion steps as mentioned before and examined by SEM. 2.9. Cytotoxicity Assay The standard methyl thiazolyltetrazolium (MTT) assay was applied to investigate the cytotoxicity of the samples. DMEM supplemented with 10 vol. % fetal bovine serum, 4.5 g L-1 glucose, and 100 units mL-1 penicillin was used to culture Murine fibroblasts cell line L929. 1 mL of medium containing the DMEM L929 fibroblasts (104 cells mL-1) was placed in each well of a 48-well plate. The plate was then incubated in a humidified 5 % CO2 in air incubator at 37 ° C for 24 h. The samples were gently placed on top of the cell layer in the well after replacing the medium with a fresh one. The control experiment was carried out using the growth culture medium without samples. After 24 h incubation at 37 °C, the culture medium and samples in 12


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each well were removed. Culture medium (900 µL) and MTT solution (5 mg mL-1 in PBS, 100 µL) were then added to each well. After 4 h incubation, the MTT solution and medium were removed and dimethyl sulfoxide (DMSO, 1 mL) was added to dissolve the formazan crystals. The optical absorbance at 490 nm was measured on a microplate reader. The results were expressed as percentages relative to the optical absorbance obtained in the control experiment. 2.10. Statistical Analysis All data were presented as mean standard deviation (SD). The statistical significance was assessed by analysis of variance (ANOVA), * (p< 0.05), ** (p < 0.01), and *** (p < 0.001). Each result was an average of at least three parallel experiments.

3. RESULTS AND DISCUSSION 3.1. Preparation of the Hierarchical Surface The SI-PIMP strategy was adopted to prepare the antibacterial hierarchical surface (Scheme 2). The silane-terminated SBDC photoiniferter was initially covalently immobilized on the silicon wafers (Si-SBDC) (Scheme S1), allowing for the subsequent living photopolymerizaiton of densely packed PSBMA bottom layer (Si-g-PSBMA). Then the Si-g-PSBMA surface was re-initiated in NIPAM/CEA aqueous solution via the living capped species at the end of PSBMA polymer chains, obtaining

the

temperature-responsive

hierarchical

surface

(Si-g-PSBMA-b-P(NIPAM-co-CEA). Antibiotic Van was covalently bonded via the 13



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reaction between its amine group and carboxyl group from CEA, endowing the surfaces with bactericidal activities.44 As reference, single-layer sample of Si-g-P(NIPAM-co-Van) was chosen to confirm the switchable properties from bactericidal to biocompatibility triggered by temperatures.

Scheme 2. The construction of hierarchical architecture via photopolymerization. 3.2. Determination of LCST of the Hierarchical Surface PNIPAM homopolymer exhibits a rapid, reversible volume phase transition at a lower critical solution temperature (LCST) of ~ 32 °C in water, along with the switchable hydration-dehydration transition.35, 36 Generally, the LCST of PNIPAM can be effectively increased by incorporation of hydrophilic charged units, such as methacrylic acid.50 In our system, comonomer CEA was chosen due to its charged property and free carboxy group for post-modification. The LCSTs of hierarchical surfaces were investigated as a function of composition ratios of NIPAM/CEA monomer mixtures. As shown in the water contact angle (WCA) test (Figure S1), increasing the CEA content (from 0 to 5 wt. %), these hierarchical surfaces showed obvious decreased WCAs when raising temperature from 25 °C to 37 °C. When the CEA content was reached 5 wt. %, the WCA differences of each samples between 25 °C and 37 °C became ambiguous, likely due to the absence of phase transition of 14


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PNIPAM, which suggested that the LCST of copolymer was higher than 37 °C.51 Moreover, to evaluate the LCST of grafted P(NIPAM-co-CEA) brushes, the random P(NIPAM-co-CEA) copolymers with different monomer weight ratios were prepared and their LCST values were tested by an UV-Vis spectrometer. Both the changes of transmittance upon temperature were simultaneously recorded, from which the midpoint temperature of the sharp transmittance was referred to as the LCST. As shown in Figure S2, the pure PNIPAM showed the lowest LCST of ~33°C, while the P(NIPAM-co-CEA)-97 exhibited an increased LCST value of ~36.5 °C because of the hydrophilic charged contribution of CEA as a comonomer. In comparison, the P(NIPAM-co-CEA)-95 with increased CEA content (5 wt. %) did not show its LCST within

the

range

of

25

°C

to

40

°C.

These

results

suggested

that

Si-g-PSBMA-b-P(NIPAM-co-CEA) with NIPAM/CEA weight ratio of 97:3 was an optimal sample in our system to exert the thermo-responsive functionality since its LCST was in the range of room temperature to physiological temperature. 3.3. Surface Characterization Both the surface wettability and thickness changes were monitored to investigate the temperature responsive behaviors of the samples. For the single-layer samples, Si-g-PNIPAM, Si-g-P(NIPAM-co-CEA) and Si-g-P(NIPAM-co-Van) were more hydrophilic at 25 °C than those at 37 °C, likely due to the hydrophilicity of PNIPAM units below its LCST (Figure 1a, b and c). On the other hand, all the hierarchical surfaces

(Si-g-PSBMA-b-PNIPAM,

Si-g-PSBMA-b-P(NIPAM-co-CEA),

Si-g-PSBMA-b-P(NIPAM-co-Van)) were more hydrophilic at 37 °C than those at 15



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25 °C when compared to the single-layer surfaces. This reversed wetting behavior confirmed the collapse of the upper PNIPAM-based copolymer chains and the domination of PSBMA at the outmost surface (Figure 1d, e and f). In addition, an exception was found on the surface of Si-g-PSBMA which showed lower contact angle at an elevated temperature (Figure 1g). To clearly illuminate the wetting behavior, water dynamic contact angles including both advancing (θa) and receding (θr) angles were investigated (Figure S3). In accordance with the results of static water contact angles, the single-layer samples (Si-g-PNIPAM, Si-g-P(NIPAM-co-CEA) and Si-g-P(NIPAM-co-Van)) were more hydrophilic both for θa and θr at 25 °C than their counterparts at 37 °C, which was likely due to the hydrophilic-hydrophobic transition of PNIPAM triggered by LCST effect. In comparison, all the hierarchical samples (Si-g-PSBMA-b-PNIPAM, Si-g-PSBMA-b-P(NIPAM-co-CEA), Si-g-PSBMA-b-P(NIPAM-co-Van)) exhibited more hydrophilic properties at 37 °C than those at 25 °C. Interestingly, at 25 °C all samples, both for one-layer and hierarchical architecture displayed nearly similar θa and θr value ranging from 54° to 61° and 22° to 31°, also revealing that the wettability of hierarchical surface was dominated by its upper layer at 25 °C. Moreover, as indicated by the layer thicknesses of samples at different temperature (25 °C and 37 °C) in Table S1, obvious decreases in layer thickness were observed on the related samples when elevating the temperature from 25 °C to 37 °C, which further confirmed the reconstruction of the grafted chains triggered by the collapse of PNIPAM segments. Among these, the hierarchical Si-g-PSBMA-b-P(NIPAM-co-Van) 16


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presented decreased layer thickness from ~39 nm to ~35 nm. The results also demonstrated indirectly the existence of the hierarchically structured block polymer brushes consisted by PSBMA and P(NIPAM-co-Van) segments. In the stability test (Figure S4), no obvious changes in WCAs were observed on both one layer and hierarchical surfaces, suggesting the resulted samples were very stable to maintain the switchable property even after 48 h immersion in PBS at 37 °C.

Figure 1. Water contact angles of the samples at 25 °C and 37 °C. (a) Si-g-PNIPAM, (b)

Si-g-P(NIPAM-co-CEA),

Si-g-PSBMA-b-PNIPAM,

(e)

(c)

Si-g-P(NIPAM-co-Van),

Si-g-PSBMA-b-P(NIPAM-co-CEA),

(d) (f)

Si-g-PSBMA-b-P(NIPAM-co-Van), (g) Si-g-PSBMA. (Error bars: standard deviation, n = 5). The expected chemical modifications on the surfaces were verified by X-ray photoelectron spectroscopy (XPS) at room temperature (Figure 2). The construction of single-layer Si-g-P(NIPAM-co-CEA) was confirmed by the characteristic peaks at 285.7 eV (C-N), 287.5 eV (O=C-N) arising from the PNIPAM, and the characteristic peaks at 286.6 eV (C-O) and 288.6 eV (O=C-O) of CEA in the high-resolution C1s spectrum. As for the hierarchical surface of Si-g-PSBMA-b-P(NIPAM-co-CEA), 17



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Page 18 of 38

besides the above-mentioned peaks for P(NIPAM-co-CEA), the predominant component at 286.2 eV originated from C-SO3-/C-N+ indicated the presence of bottom PSBMA layer. After Van immobilization, new peak at 202.3 eV (Wide-scan XPS spectra,

Figure

S5)

with

Si-g-P(NIPAM-co-Van)

and

respect

to

the

Cl2p

was

observed

Si-g-PSBMA-b-P(NIPAM-co-Van)

with

on

both atomic

concentrations of 1.00 % and 1.22 %, respectively (Table S2).

(a)

(b)

(d)

(c)

(e)

(f)

Figure 2. C1s core-level XPS spectra at room temperature for (a) Si-SBDC, (b) Si-g-P(NIPAM-co-CEA),

(c)


(d)

Si-g-PSBMA,

(e)

Si-g-PSBMA-b-P(NIPAM-co-CEA), (f) Si-g-PSBMA-b-P(NIPAM-co-Van). 3.4. Bacterial Killing and Resistance Induced by Temperature Biomedical devices might be affected by ubiquitously bacterial contamination during storage period, causing biofilm formation, device-associated infections, even 18


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functional failure. Hence, devices exhibiting bactericidal properties during storage at room temperature and turnable bacterial repellency and biocompatible properties at physiological temperature are highly desired. As a powerful glycopeptide antibiotic, Van could kill a wide range of Gram-positive bacteria by interfering with the biosynthesis of bacterial cell walls, but could not penetrate to Gram-negative pathogens because of an additional outer membrane.48 Therefore, Gram-positive S. aureus

was

chosen

as

representative

microorganism

to

examine

the

temperature-triggered switchable antibacterial performances, with the Gram-negative E. coli as a control. Experimentally, the samples were incubated in 1 mL of S. aureus and E. coli suspensions (107 cells mL-1) at given temperatures (25 °C and 37 °C) for 6 h, bacterial viability was tested via standard fluorescent LIVE/DEAD BacLight kit (propidium iodide (PI)/SYTO-9). In this stain assay, the SYTO-9 can label all bacteria with green fluorescence; while PI only penetrates bacteria with damaged membranes, displacing the green fluorescence with red. Therefore, viable bacterial cells show green fluorescence while dead bacterial cells are in red color. As shown in the representative CLSM images (Figure 3A and Figure S7A), at room temperature (25 °C), quite a number of viable S. aureus and E. coli with green fluorescence

adhered

on

the

control

Si

surfaces.

For

the

samples

of

Si-g-PSBMA-b-P(NIPAM-co-Van) and Si-g-P(NIPAM-co-Van), a great number of viable E. coli (green spots) were found on the surfaces, which was in accordance with the previous result that Van was failed to present antibacterial property against E. coli (Figure S7). In stark contrast, most adherent S. aureus on those two surfaces were 19



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dead, as indicated by a large number of red spots, showing the selective bactericidal property. Among these, Si-g-PSBMA-b-P(NIPAM-co-Van) showed a killing efficiency of ~ 88.6 %, comparable to its counterpart of Si-g-P(NIPAM-co-Van) (~ 91.7 %) (Figure 4). These results demonstrated that the Van moieties, in the stretchable upper layer, were accessible to contact with bacterial cells and exert biocidal performance at 25 °C. While at physiological temperature (~37 °C), Si-g-PSBMA-b-P(NIPAM-co-Van) showed the significant decreases in both fluorescence intensity and bacterial coverage for both S. aureus and E. coli as compared to that of Si-g-P(NIPAM-co-Van), revealing the surface possessed remarkable anti-adhesion property (Figure 3B and Figure S7B). Moreover, Si-g-PSBMA-b-P(NIPAM-co-Van) showed an even lower killing efficiency (~0.8 %) against S. aureus than that of Si-g-P(NIPAM-co-Van) (~17.1 %), further demonstrating that the surface properties of hierarchical surface had changed from bactericidal to dominantly anti-adhesive triggered by the elevated temperature (Figure 4, Figure S6 and Figure S8). The effective antibacterial adhesion and depressed bactericidal performances of the hierarchical surface were mainly attributed to the collapsed P(NIPAM-co-Van) upper layer, which blocked the interactions between the Van groups and bacterial cells, facilitated the exposure of underlying PSBMA bottom layer. Meanwhile, driven by the reorganized hydrophobic/hydrophilic balance at 37 °C (PSBMA was more hydrophilic and PNIPAM was relatively hydrophobic at this temperature), the hydrophilic PSBMA in hierarchical surface could better conceal the coiled hydrophobic PNIPAM, as well as 20


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the Van, thus largely inhibiting the bactericidal activity.

A 25°C

B 37°C

(a)

(b)

(c)

(d)

Figure 3. Representative CLSM images of S. aureus attached on samples. The samples were exposed to bacterial suspension (107 cells mL-1) in buffers for 6 h at 25

°C

(A)

and

37

°C

(B),

(a)

Si,

(b)


(c)

Si-g-PSBMA-b-P(NIPAM-co-Van), (d) Si-g-PSBMA. (Scale bar is 50 µm).

Figure 4. The killing efficiencies after 6 h of incubation in S. aureus suspension at 25

°C

and

37

°C.

(a)

Si,

(b)


(c)

Si-g-PSBMA-b-P(NIPAM-co-Van), (d) Si-g-PSBMA. Significant difference (* p < 0.05, ** p < 0.01 and *** p < 0.001). (Error bars: standard deviation, n = 3). 21



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Additionally, the morphologies of the adhered S. aureus at 25 °C and 37 °C were examined by SEM (Figure 5A and 5B). Large numbers of S. aureus cells were observed

on

the

surfaces

of

both

Si-g-P(NIPAM-co-Van)

and

Si-g-PSBMA-b-P(NIPAM-co-Van) at 25 °C, which appeared to be shrunk and fused and sharply contrast to the ball-shaped cells on the control Si and Si-g-PSBMA, suggesting the leakage of cytoplasm and obvious loss of cellular integrity.52 At 37 °C, Si-g-PSBMA-b-P(NIPAM-co-Van) exhibited evident reduction of bacteria adhesion than Si-g-P(NIPAM-co-Van). Notably, the adhered bacteria on hierarchical surface retained their smooth and regular ball-shape; whereas there were still a large number of dead bacteria attached on the single-layer Si-g-P(NIPAM-co-Van) surface since not all Van groups were buried and loss their bactericidal activity. These results further confirmed that bottom PSBMA layer played a predominant role in realizing the temperature-triggered switchable properties from bactericidal to bacterial repellency.

A 25°C

B 37°C

(a)

(b)

(c)

(d)

Figure 5. Representative SEM images of S. aureus attached on samples. The samples were exposed to bacterial suspension (107 cells mL-1) in buffers for 6 h at 25 °C (A) and

37

°C

(B),

(a)

Si,

(b)


22


(c)

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Si-g-PSBMA-b-P(NIPAM-co-Van), (d) Si-g-PSBMA. 3.5. Bacterial Detachment Traditional contact-killing antibacterial surfaces often suffer from severe dead bacteria adhesion, which would compromise the antibacterial activity and facilitate subsequent bacterial adhesion for a new burst of bacterial invasion. Herein, to test the bacteria releasing capability triggered by physiological temperature, the samples that previously attached/killed bacteria were gently washed with buffers at 37 °C for 1 h. The number of remained S. aureus on surfaces before and after washing was recorded by both CLSM and SEM (Figure 6 and Figure 7). It was found that Si-g-PSBMA-b-P(NIPAM-co-Van) could release ~ 72.5 % of previously attached dead

S.

aureus,

showing

“self-cleaning”

performance.

In

contrast,

Si-g-P(NIPAM-co-Van) exhibited no obvious bacteria detachment after the washing process, as indicated by a large number of dead bacteria and unchanged bacterial coverage. A similar tendency was also observed on the E. coli detachment test (Figure S9 and Figure S10). The adhered bacteria on Si-g-PSBMA-b-P(NIPAM-co-Van) surfaces were released obviously, while the one-layer Si-g-P(NIPAM-co-Van) samples still attached a large number of bacteria. We speculated that synergistic effects of two factors were responsible for the superior performances of bacteria release on the Si-g-PSBMA-b-P(NIPAM-co-Van) hierarchical surface. First, the collapsed PNIPAM chains played an important role in disrupting the primary hydrogen bonding between the Van and bacteria; meanwhile, the hydration of PSBMA brushes further undermined the aforementioned hydrogen bonding. Importantly, due to the 23



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Page 24 of 38

configuration rearrangement of the polymer brushes to decrease the free energy of the hierarchical system, the hydrophilic PSBMA segments substantially appeared at the outermost surface, whereas the relatively hydrophobic P(NIPAM-co-Van) tended to migrate towards inner surface, significantly benefiting the releasing of attached bacteria.49 Compared with the previous report that release of bacterial residuals from a biocidal surface via additional chemical treatment,26 our strategy solely utilized the elevated temperature as the trigger for a conformational rearrangement on the hierarchical surface.

A

B

(a)

(b)

(c)

(d)

Figure 6. Representative CLSM images of S. aureus attached onto samples before and after washing. The samples were exposed to bacterial suspension (107 cells mL-1) in buffers at 25 °C for 6 h (A) and then washed with 37 °C buffers in a shaker for 1 h (B). (a) Si, (b) Si-g-P(NIPAM-co-Van), (c) Si-g-PSBMA-b-P(NIPAM-co-Van), (d) Si-g-PSBMA. (Scale bar is 50 µm).

24


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Figure 7. The attachment and detachment of S. aureus on samples. The samples were exposed to bacterial suspension (107 cells mL-1) in buffers at 25 °C for 6 h and the average number of attached cells was counted from representative SEM images. The samples were then washed with 37 °C buffers in a shaker for 1 h, and the remaining cells were counted. The corresponding release ratio was also shown. (a) Si, (b) Si-g-P(NIPAM-co-Van), (c) Si-g-PSBMA-b-P(NIPAM-co-Van), (d) Si-g-PSBMA. (Error bars: standard deviation, n = 3). 3.6. Blood Cell Adhesion One major concern for implant materials is about its biocompatibility, which is critical for appropriate application in vivo.53 To qualitatively evaluate surface biocompatibility, the adhesion of platelets and erythrocytes on the samples at 37 °C were investigated by SEM (Figure 8). In the case of pristine Si and Si-g-P(NIPAM-co-Van), a large number of adhered platelets with the elongated pseudopodia and spreading morphology were found on surfaces, indicating these surfaces easily triggered a lot of platelets adhesion and activation. In contrast, only a few platelets in round or disc-like shape (nonactivated state) were observed on the Si-g-PSBMA-b-P(NIPAM-co-Van) surface, which was 25



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Page 26 of 38

comparable to those on the Si-g-PSBMA surface. The results demonstrated that Si-g-PSBMA-b-P(NIPAM-co-Van) surface exhibited obvious anti-platelet adhesion properties due to the domination of PSBMA at the outmost surface, which was caused by the temperature-triggered configurational change on hierarchical surface. As for erythrocyte assay (Figure 8B), large numbers of erythrocytes were found on pristine Si and Si-g-P(NIPAM-co-Van), nearly all adhered erythrocytes showed activated state with the cell deformation and lysis. In stark contrast, the single layer Si-g-PSBMA showed extremely low adhesion of disc-like erythrocytes, consisting well with the previous work that PSBMA modified surface revealed excellent biocompatibility

with

erythrocytes.54

On

the

other

hand,

the

Si-g-PSBMA-b-P(NIPAM-co-Van) exhibited extremely low erythrocytes adhesion with only few cells activated. These results suggested that this switchable hierarchical surface at 37 °C would possess good biocompatibility to blood cells.

A

B

(a)

(b)

(c)

(d)

Figure 8. Representative SEM images of platelets (A) and erythrocytes (B) upon contact with surfaces at 37 °C. (a) Si, (b) Si-g-P(NIPAM-co-Van), (c) Si-g-PSBMA-b-P(NIPAM-co-Van), (d) Si-g-PSBMA. (The insets in all pictures are 26


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magnified views of adhered blood cells with different activation levels.) 3.7. Cytocompatibility Assay The cytotoxicity of the as-prepared samples was evaluated by an MTT viability assay against L929 cells line at 37 °C incubation. After 24 h of cell-surface interaction, cell viabilities on all sample surfaces were higher than 90 % and no statistic differences in the cell viability were found among these samples (Figure 9), implying that the Si-g-PSBMA-b-P(NIPAM-co-Van) had no adverse effects on the mammalian cells.

Figure 9. Viability of L929 fibroblasts on samples at 37 °C. (a) Si, (b) Si-g-P(NIPAM-co-Van), (c) Si-g-PSBMA-b-P(NIPAM-co-Van), (d) Si-g-PSBMA. (nsd = no significant difference (p > 0.05)). (Error bars: standard deviation, n = 3).

4. CONCLUSION In

summary,

a

novel

temperature-responsive

hierarchical

surface

that

synergistically integrated bactericidal upper layer and antifouling zwitterionic bottom layer had been presented. The bactericidal experiments demonstrated that the temperature-triggered hydration and conformational changes of hierarchical surface 27



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could modulate the spatial exposure and concealment of Van. The hierarchical surface could effectively kill bacterial cells at room temperature and automatically switch to a zwitterionic bacterial repellency surface at physiological temperature, accompanied by the release of dead bacterial residues and inhibition subsequent attachment of planktonic bacteria. Moreover, the exposure PSBMA layer conferred the surface better biocompatibility. The proof-of-concept demonstration of hierarchical surface with switchable bioactivity could be advantageously implemented by integrating various antifouling materials and bactericidal agents with PNIPAM, which will be greatly attractive for preparing the infection-resistant medical devices.

28


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ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website http://pubs.acs.org. Schematic of the immobilization of disulfide-containing photoiniferter, water contact angles of the hierarchical surfaces with varied feed ratios of NIPAM and CEA (wt. %), LCSTs of NIPAM based random copolymers, advancing and receding contact angles of the samples at 25 °C and 37 °C, thickness and graft density of the samples, the stability of samples, wide-scan XPS spectra, quantitative XPS data, statistical analysis of the percentage surface coverage of S. aureus at 25 °C and 37 °C, representative CLSM images of E. coli attached on samples at 25 °C and 37 °C, statistical analysis of the percentage surface coverage of E. coli at 25 °C and 37 °C, representative CLSM images of E. coli attached on samples before and after washing, statistical analysis of the percentage surface coverage of E. coli before and after washing (PDF)

AUTHOR INFORMATION Corresponding Authors *Tel.: +86 431 85262161; Fax: +86 431 85262109. E-mail: [email protected]. *E-mail: [email protected]. Notes The authors declare no competing financial interest. 29



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ACKNOWLEDGMENTS The authors acknowledge the ﬁnancial support of the National Natural Science Foundation of China (Project Numbers: 51473167), Scientific Development Program of Jilin Province (Project Numbers: 20170520123JH), and Chinese Academy of Sciences-Wego Group High-Tech Research & Development Program.

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320x280mm (48 x 48 DPI)


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Temperature-Responsive Hierarchical Polymer Brushes Switching

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