Substrate-Independent Ag-Nanoparticle-Loaded Hydrogel Coating

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Substrate independent Ag nanoparticles loaded hydrogel coating with regenerable bactericidal and thermo-responsive anti-bacterial properties Min He, Qian Wang, Jue Zhang, Weifeng Zhao, and Changsheng Zhao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b13238 • Publication Date (Web): 16 Oct 2017 Downloaded from http://pubs.acs.org on October 17, 2017

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ACS Applied Materials & Interfaces

Substrate independent Ag nanoparticles loaded hydrogel coating with regenerable bactericidal and thermo-responsive anti-bacterial properties Min He, Qian Wang, Jue Zhang, Weifeng, Zhao* and Changsheng, Zhao*

College of Polymer Science and Engineering, State Key Laboratory of Polymer Materials Engineering, Sichuan University, Chengdu 610065, People’s Republic of China * Corresponding author. Tel: +86-28-85400453, Fax: +86-28-85405402, E-mail: [email protected] or [email protected] (W.F. Zhao); [email protected] or [email protected] (C.S. Zhao)

ACS Paragon Plus Environment


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Abstract: We report an Ag nanoparticles (AgNPs) based substrate independent bactericidal hydrogel coating with thermo-responsive anti-bacterial property. To attach the hydrogel coating onto model substrate, ene-functionalized dopamine was firstly coated on the substrate, and then the hydrogel thin layer was formed on the surface via the UV light initiated surface cross-linking copolymerization of N-isopropylacrylamide (NIPAAm) and sodium acrylate (AANa). Then, Ag ions were adsorbed into the hydrogel layers and reduced to AgNPs by sodium borohydride. The coating showed robust bactericidal ability against Escherichia coli and Staphylococcus aureus toward both contacted bacteria and the bacteria in the surrounding. Upon a reduction of the temperature below the LCST of PNIPAAm, the improved surface hydrophilicity and swollen PNIPAAm could detach the attached dead bacteria. Meanwhile, the long-lasting and regenerable antibacterial properties could be achieved by repeatedly loading AgNPs. By precisely controlling the AgNPs loading amounts, the coating showed excellent hemocompatibility and no cytotoxity. Additionally, the coating could be applied to modify cell culture plate, since it could support cell adhesion and proliferation at 37 °C, while detach the cell by changing the temperature below lower critical solution temperature without the treatment of proteases. The study thus presents a promising way to fabricate thermo-responsive and regenerable antibacterial surfaces on diverse materials and devices for biomedical applications. Key words: thermo-responsive, antibacterial surface, hydrogel thin layer, intelligent surface, Ag nanoparticles.


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1. Introduction Biomaterials exposed to nonsterile aqueous environment quickly become the supports for attaching bacteria, which further elaborate into three-dimensional biofilms.1 For medical devices, bacteria infections adversely affect the functionality and limit the lifetime of devices, and also represent a common and substantial complication in clinical practice.2 Each year in the USA alone, bacteria infections lead to the death of more than one million people. In Europe, hospital-acquired bacteria infections also bring enormous suffering to more than 5 % of patients and the mortality is about 10 % per year.3 To solve the problems, considerable efforts have been paid to design antibacterial surfaces that can greatly reduce the extent of initial bacterial attachment and thereby prevent subsequent biofilm formation.4-5 In recent decades, there are two main categories of antibacterial surfaces: bactericidal surfaces for killing attached bacteria or the bacteria in the surrounding using components such as quaternary ammonium salts (QAS), chitosan, or silver ions;6 bacterial-resistant surfaces for preventing bacterial attachment based on hydrophilic coatings, such as poly(ethylene glycol), poly(vinylpyrrolidone), and zwitterionic polymers.7-8 Although remarkable progress has been acheived in the development of these antibacterial surfaces, each category has its inherent advantages and disadvantages. For bactericidal surfaces, they can prevent the formation of biofilms for a certain period of time, but dead bacteria can easily accumulate on the surfaces, which would promote further bacterial contamination and finally lead to the formation of biofilms.9-10 Additionally, as the most widely used biocides to prepare antibacterial surfaces,11-13 QAS and AgNPs are capable of effectively killing bacteria.14-15 However, the QAS based antibacterial surfaces can only target on the bacteria contacting with the surface, having no influence on the bacteria reproduction in the surrounding;16 while the AgNPs would be finally consumed out, so the antibacterial property can be only maintained for a period of time.17 On the other hand, bacteria-resistant surfaces can inhibit the initial attachment of bacteria; however, no such surfaces can completely resist the contamination of many kinds of proteins such as fibronectin, fibrin, and fibrinogen, which would promote bacterial adhesion by providing donors, inevitably becoming colonized by bacteria.18 Consequently, an ideal antibacterial surface should be capable of preventing initial bacterial attachment, subsequently killing all attached bacteria, and finally removing dead bacteria.19 To achieve this goal, researchers have focused their attentions on designing and constructing environment responsive antibacterial surfaces, such as 20 21 22-23 24 thermo-responsive, pH-responsive, enzyme-responsive, photoresponsive, and so on. Poly(N-isopropylacrylamide) (PNIPAAm) based thermal responsive surfaces are a kind of prototypical smart surfaces, which display a sharp, reversible solubility phase transition at a lower critical solution temperature (LCST) of 32 °C.25 As a result, PNIPAAm-modified surfaces exhibit switchable wettability and bioadhesion property, and the property has been exploited as a model fouling-release material.26-27 Many researchers have integrated biocides with PNIPAAm in the form of brushes or



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nanopatterned brushes to achieve desired antibacterial surfaces with the capacity of controllable attaching the bacteria, killing the bacteria, and detaching dead bacteria.28 For example, Yu et al. constructed nanopatterned PNIPAAm brushes on silicon wafers, and two common biocides of quaternary ammonium salt (QAS) and lysozyme are respectively immobilized in polymer-free regions.29-30 Shi et al. developed a facile method to construct a thermo-responsive antibacterial surface on silicon wafer by host-guest self-assembly of β-cyclodextrin with PNIPAAm terminated adamantine and poly[2-(methacryloyloxy)ethyl]trimethylammonium chloride terminated 31 adamantine. Although these thermo-responsive antibacterial surfaces effectively exhibited both biocidal and dead bacteria detaching functionalities, the constructing of these surfaces involved multiple steps, reguired complex equipment and rigorous condition, and might not be suitable for other non-silica-based substrates, which limited their broad applications. In our previous works, we developed a simple technique to covalently attach functional hydrogel thin layers onto polyethersulfone (PES) membrane surface.7 With the technique, super-anticoagulant PES surface and robust antibacterial surface were prepared.32-33 In our strategy, double bond grafted PES membrane was firstly prepared via in situ crosslinking polymerization of hydroxyethyl methylacrylate (HEMA) in PES solution (followed by the reaction between the hydroxyl groups and acryloyl chloride), then the hydrogel thin layers were formed and covalently attached onto PES membanes by an UV-light initiated surface copolymerization. However, the method of introducing anchoring points for hydrogel layer was also not versatile for other substrates, and the lifetime of the prepared antibacterial hydrogel coating was limited for the using up of AgNPs and the contamination of the dead bacteria. To overcome these drawbacks, we anticipated to develop a substrate independent antibacterial hydrogel coating with regenerable long-lasting antibacterial and thermo-responsive bacteria detachmennt properties. To achieve these goals, in-situ crosslinking of HEMA was replaced by coating ene-functionalized dopamine to introduce anchoring sites for hydrogel layer, which was able to closely attach onto almost all kinds of substrates. NIPAAm and sodium acrylate (AANa) were selected as functional monomers to form hydrogel layer. The AANa segments were used to adsorb Ag ions, and could be repeatedly used to regenerate the biocidal efficacy, while the NIPAAm segments could provide thermo-responsive bacterial detachment property. In our strategy, double bond grafted dopamine was firstly synthesized via the reaction of the amino group with acryloyl chloride, then coated onto a model substrate to introduce double bonds as the anchoring sites. Sequentially, the hydrogel coating was formed via the surface free radical crosslinking copolymerization of NIPAAm and AANa, followed by adsorbing Ag ions and reduced by sodium borohydride. The surface morphology and chemical compositions were investigated by scanning electron microscopy (SEM), attenuated total reflection-Fourier transform infrared (ATR-FTIR), X-ray diffraction (XRD), and EDS. The thermo-responsive hydrophobic/hydrophilic property was evaluated by water contact angle and swelling degree at 37 °C and 4 °C, respectively. The


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bactericidal and thermo-responsive bacteria detachment properties were characterized by optical degree of co-culture solution, inhibition zone, and live/dead staining methods. The hemocompatibility and cytocompatibility of the hydrogel layer were also evaluated by hemolysis and cytotoxity, respectively. Additionally, the thermo-responsive cell detachment property was also investigated.



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2. Materials and experiments 2.1 Materials Commercial polyethersulfone (PES) was purchased from BASF. Sodium acrylate (AANa) (98%), N-isopropylacrylamide (NIPAAm), 2-ketoglutaric acid (KTA) (99%), and N, N’-methylenebisacrylamide (MBA) (99%) were purchased from Aladdin Chemistry Co. Ltd. N-methyl-2-pyrrolidinone (NMP) (AR), silver nitrate, dopamine, and sodium borohydride were purchased from Chengdu Kelong Inc. (Chengdu, China), and used as received. Triethylamine (98%) and acryloyl chloride (99%) were purchased from Best-reagent Ins. All the other chemical regents were obtained from Chengdu Kelong Inc. (Chengdu, China), and were used without further purification. Dulbecco’s modified eagle medium (DMEM), fetal bovine serum (FBS), 0.05 % Trypsin EDTA, CCK-8 agent, MTT agent, and phosphate buffer saline (PBS, pH=7.4) were purchased from Gibco (USA). Deionized (DI) water was used throughout the experiments. 2.2 Immobilization Ag nanoparticles (AgNPs) loaded hydrogel coating onto substrate The AgNPs loaded hydrogel coatings were synthesized via three steps: (i) ene-functionalized dopamine was synthesized, and then coated onto PES membrane surface to introduce double bonds; (ii) thermo-responsive hydrogel layers were formed and attached onto the membrane surface via surface cross-linking copolymerization; (iii) Ag ions were adsorbed into the hydrogel layer and reduced to AgNPs. The details were provided in the Supporting Information. 2.3 Physical and chemical characterizations of hydrogel thin layer attached membranes The morphology and chemical compositions were characterized by scanning electron microscopy (FE-SEM), attenuated total reflection-Fourier transform infrared (ATR-FTIR), EDS maping, and X-ray diffraction (XRD). The surface hydrophilicity of the membranes at 37 and 4 °C were investigated on the basis of contact angle measurement, using a contact angle goniometer (OCA20, Dataphysics, Germany) equipped with a video capture. The swelling behaviors at 37 and 4 °C were also investigated. The details were provided in the Supporting Information. 2.4 Total AgNPs content and release behavior tests The total AgNPs loading amounts and the release behavior of the loaded AgNPs were tested by inductively coupled plasma-mass spectrometry (ICP-MS). The details were provided in the Supporting Information. 2.5 Antibacterial activity tests Escherichia coli (E. coli, gram negative) and Staphylococcus aureus (S. aureus, gram positive) were used as the model bacteria to evaluate the bactericidal and


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thermo-responsive bacteria detachment properties of the AgNPs loaded hydrogel thin layers by the optical degree of co-culture solution, inhibition zone, and live/dead two color fluorescene methods. The details were provided in the Supporting Information. 2.6 Biocompatibility tests Hemolysis and cytotoxity were tested to evaluate the biocompatibility of the AgNPs loaded hydrogel coating. Additionally, the thermo-responsive cell detachment property of the hydrogel layer was also investigated with L929 cell as model. The details were provided in the Supporting Information.



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3. Results and discussion 3.1 Morphologies, chemical properties of the membranes

compositions,

and

physicochemical

To covalently attach a hydrogel thin layer onto a substrate, the key point is to create anchoring sites for the hydrogel layer.34-35 Many researchers have introduced functional groups as anchoring sites onto silica-based substrates to immobilize hydrogel thin layers via the reaction of different silane coupling agents.36-38 In our previous studies, in situ crosslinking copolymerization of HEMA was carried out to introduce hydroxy groups onto PES membrane as anchoring sites.7, 32 However, for these techniques, special chemical structures of substrates are prerequisite, limiting their wide applications. In this study, ene-functionalized dopamine was used to introduce double bonds onto substrate surfaces as anchoring sites for hydrogel layers, which was versitile for various substrates, such as polymers, metals, and inorganic materials.39-40 Many studies have reported the synthesis of ene-functionalized dopamine in the protonic solvent, and the hydroxyl groups of dopamine needed to be protected in these systems.41 However, we synthesized ene-functionalized dopamine via the reaction of acryloyl chloride and dopamine in the NMP solution. The NMP was a non-protonic solvent, and the hydroxyl groups of dopamine would not ionize protons in the system, so there was no necessity to protect the hydroxyl groups. AgNPs losded hydrogel coating was fabricated via three steps, as shown in Scheme 1: introducing double bonds via the coating of ene-functinoalized dopamine; forming and attaching of the hydrogel coating via surface crosslinking copolymerization of NIPAAm and AANa under UV light at 365 nm; loading AgNPs into the coating. The surface morphology, chemical compositions, and hydrophilicity at 4 and 37 °C of the coating were characterized by SEM, ATR-FTIR, XRD, EDS, water contact angle, and swelling degree.

Scheme 1. The process of chemically attaching hydrogel thin layer onto PES membrane.


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Figure 1. Surface morphologies of the pure and hydrogel layers attached membranes.

The surface morphologies of pure PES and modified PES membranes were observed by SEM, as shown in Figure 1. The pure PES membrane showed a flatten morphology with uniformly distributed pores, which were formed during the phase inversion process. After coating dopamine, the pores were covered by the dopamine coating layer, displaying a smooth surface morphology. For the hydrogel layer attached membranes, rough and honeycomb-like porous surface morphologies were observed, and the pore size showed an increasing tendency with the increase of the AANa proportions in the hydrogel layers, which might be ascribed to the charge repulsion of the carboxylate groups. It was noteworthy that the typical porous hydrogel structure could only be observed when the sample was freeze-dried (to maintain its porous structure) and observed immediately, for the reason of easily shrinking of the hydrogel thin layer.

Figure 2. (A) ATR-FTIR spectrum of the pure PES membrane and modified PES membrane; (B) XRD spectrum of the AgNPs loaded hydrogel layers.

The surface chemical compositions were characterized by ATR-FTIR, as shown in Figure 2(A). Compared with the spectrum of pure PES membrane, the emerged new peak at 1650 cm-1 on PES-DA membrane was assigned to the characteristic peak of the amide bond, coming from the double bond grafted dopamine. After attaching the



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hydrogel layer, the characteristic peak of amide bond was enhanced by the amide bond of NIPAAm. The new emerged peak at 1720 cm-1 was assigned to the streching vibrations of the carboxylate groups of AANa, and the intensity increased with the increase of the AANa proportions in the hydrogel layers. The results further confirmed that the hydrogel layers were sucessfully attached onto the membrane surfaces. The presence of AgNPs in the hydrogel layers was analyzed by XRD, as shown in Figure 2(B). Compared with PES-DA, the emerged diffraction peaks at 38.5°, 44.8°, 64.7° and 77.8° were assigned to the (111), (200), (220) and (311) crystalline planes of AgNPs, respectively,42 indicating the presence of AgNPs in the coatings. Additionally, the mean diameter of the AgNPs was about 108.7 nm analyzed by nano measurement software, as shown in Figure 3.

Figure 3. The mean diameter of AgNPs.

Figure 4. AgNPs distribution in the hydrogel layers analyzed by EDS-mapping.

EDS-mapping was further used to qualitatively analyze the AgNPs loading amounts and distribution, as shown in Figure 4. The green dots represented the AgNPs, the density and intersity of the green dots qualitatively represented the AgNPs loading amounts. With the increase of the AANa proportions in the hydrogel layers, the green dot density increased, and the green light intensity was enhanced, indicating that the AgNPs loading amounts increased with the increase of the AANa proportions in the hydrogel layers. Additionally, some locations displayed green lightspots, indicating the aggrgation of the AgNPs, but most of the AgNPs was uniformly distributed in the hydrogel layers. Additionally, the catechol group of dopamine could also reduce the Ag+ to AgNPs,17 so the AgNPs could also locate at the interface of dopamine coating layer and the hydrogel layer. As demonstrated above, the AgNPs loaded hydrogel layers were successfully attached onto the substrate surface. Considering the actual applications of the hydrogel layer, the hydrogel layer thickness and the stability of the hydrogel layer were two key factors. In this study, the hydrogel layers were formed between two quartz glass sheets; so the thickness of the hydrogel layers could be well-controlled by regulating the spacer thickness or the concentrations of the hydrogel precursors. The dependence of the thickness on hydrogel precursor concentrations with fixed spacer thickness was studied by the cross sectional SEM images (images not shown). It was


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found that the thicknesses of the hydrogel thin layers increased with the increase of the hydrogel precursor concentrations (Figure S1). The thinnest thickness of the layer was about 0.61 µm, since the surface polymerization could hardly be initiated once the precursor concentration was too low. The delamination of the attached hydrogel layers was a great threaten for long-term applications; thus, the stability of the layers was evaluated by calculating the residual masses of the samples after each cycle of immersing-drying treatment. The dried sample of PES-85-15 was immersed into pure water for 12 h, and then dried at room temperature. Both the swelling state and dried state masses of the membrane were weighted. The immersing-drying treatment was repeated for seven cycles, and no obvious mass change was observed (Figure S1(B)). 3.2 Thermo-responsive switchable surface hydrophilicity Recently, a class of thermo-responsive wettability of PNIPAAm modified surfaces were prepared, and generated great interest in the biomaterials community.24, 43 The sharp transition between super-hydrophilicity and super-hydrophobicity was mainly ascribled to the competition between intermolecular and intramolecular hydrogen bonding below and above LCST. Below LCST, the PNIPAAm polymer is extended, and in solvent-swelled conformation; but when heated up above LCST, the polymer undergoes a phase transition to yield a collapsed morphology, and leads to the exclusion of solvent. To evaluate the thermo-responsive surface property, the wettability of the hydrogel layers at 4 and 37 °C was tested by water contact angle (WCA), as shown in Figure 5(A). The WCA values of the PES and PES-DA membranes barely changed at the two temperatures, whereas the hydrogel coating attached membranes exhibited temperature-responsiveness, and the WCA decreased from around 80° at 37 °C to around 55° at 4 °C. Additionally, the WCAs of hydrogel layer attached membranes were slightly higher than the pure PES membrane, which should be ascribed to the improved surface roughness. To further confirm the thermo-responsiveness property, the swelling degrees of the hydrogel coatings at 4 and 37 °C were also investigated, as shown in Figure 5(B). The hydrogel layers showed obvious higher swelling degree at 4 °C. The results demonstrated that the hydrogel layers retained the thermo-responsive wettability. Additionally, with the increase of the AANa proportions in the hydrogel layers, the WCA slightly decreased, and the swelling degree slightly increased, indicating that the AANa segments could increase the hydrophilicity of the hydrogel layers.

Figure 5. (A) The water contact angles of the membrnes at 4 and 37 °C; (B) the swelling degree of the attached hydrogel layers at 4 and 37 °C.



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3.3 The loading (reloading) and releasing of AgNPs AgNPs are widely used to prepare antibacterial materials or surfaces because of their strong and broad-spectrum antibacterial characteristics.44-45 The main mechanism of killing bacteria is to release Ag+ ions to damage bacterial membrane as well as disrupt the function of bacterial enzymes and nucleic acid groups in cellular protein and DNA.19 However, it was reported that AgNPs would cause cytotoxicity and severe hemolysis when the loading amounts surpassed a certain threshold,33 so the AgNPs loading amounts must be controlled below the threshold by reducing the concentration of silver nitrate solution and the adsorption time, as well as the proportions of the carboxylate groups in the hydrogel layers. In this study, the AgNPs loading amounts of PES-90-10, PES-85-15, PES-80-20, and PES-75-25 were 5.1, 8.8, 13.5, and 18.3 µg/cm2, respectively (Figure 6(A)). For AgNPs based antibacterial materials, the main drawback was that the antibacterial property could only be maintained for a period of time for the consuming of AgNPs. For the PES-90-10 and PES-85-15, the AgNPs releasing could last for about 5 days, where the PES-80-20 and PES-75-25 could last for more than 8 days, as shown in Figure 6(B). To provide long-term and regenerable antibacterial property, the carboxylate groups in the hydrogel layers were designed to reload AgNPs. As shown in Figure 6(A), the modified membranes after using up AgNPs were used to reload AgNPs, and the membranes showed the same loading capacities even after three releasing-reloading cycles.

Figure 6. (A) AgNPs loading amounts for the modified membranes after each cycle reloading; (B) the releasing profile of AgNPs vs time; (C) the optical degrees of AgNPs loaded membranes for E. coli and S. aureus; (D) the optical degrees of PES-85-15 after different releasing time and several


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releasing-reloading cycles.

3.4 Bactericidal and thermo-responsive bacteria detachment properties The loaded AgNPs could be released into the medium in the form of Ag+ ions to kill bacteria and inhibit bacteria growth. The optical degree of co-culture solutions with E. coli and S. aureus were firstly used to evaluate the antibacterial activity of the modified membranes, as shown in Figure 6(C). PES and PES-DA showed O.D. values of around 0.45, displaying no antibacterial property. For all the AgNPs loaded membranes, robust bactericidal activities were demonstrated by the dramatically decrease of O.D. values to around 0. The releasing based antibacterial property of the membranes was also visually investigated by the inhibition zone, as shown in Figure 7. No distinct margins were observed for the PES and PES-DA, while obvious inhibition zones were observed for all the AgNPs loaded membranes for the releasing of AgNPs. To confirm the long-term and regenerable antibacterial properties, the sample of PES-85-15 after releasing Ag+ for 1, 2, and 3 days were used to measure the optical degree of the coculture solutions with E. coli. As shown in Figure 6(D), the O.D. values increased with the increase of releasing time, indicating that the antibacterial activity decreased along with the consuming of the AgNPs. However, after reloading AgNPs, the membranes showed robust antibacterial property again, indicating that the robust and long-term antibacterial property could be provided by reloading AgNPs.

Figure 7. The inhibition zone images of the modified membranes towards E. coli and S. aureus.

Above analysis demonstrated that the modified membranes showed robust and regenerable bactericidal property. However, for most bactericidal surfaces, the main drawback was that the dead bacteria would also contaminate the surface, and finally lead to the formation of biofilms.46 To investigate the thermo-responsive bacteria detachment property of the hydrogel layer, the bacteria co-cultured membranes were immersed into PBS solution at 37 and 4 °C for 30 min, respectively. Then, the rinsed membranes were stained by FDA/PI before being observed by fluorescence microscope, and the results were shown in Figure 8. For the PES and PES-DA, large sums of live bacteria were observed on the surfaces, and the temperature showed little



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influence on the number of the attached bacteria. For pure NIPAAm hydrogel layer attached membrane, the number of attached live bacteria decreased obviously after immersing and rinsing in 4 °C PBS solution. For the AgNPs loaded hydrogel layer attached membranes, a few live bacteria and large sums of dead bacteria were observed on the surfaces at 37 °C. However, after immersing into 4 °C PBS solution, most bacteria were detached from the surfaces. The results indicated that the designed surfaces could effectively kill and detach the bacteria in response to the temperature change to avoid the contamination of the membrane surface. The mechanism of detaching the attached bacteria was ascribed to the increased surface hydrophilicity and the increased swelling ratio at 4 °C.


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Figure 8. The fluorescence images of the bacteria cocultured membranes after immersed and rinsed at 37 °C and 4 °C, respectively.

3.4 Hemolysis and cytotoxity tests. The designed AgNPs loaded hydrogel coating was substrate independent for that the anchoring sites of the coating was created by a mussel-inspired method, which meant that the coating could be applied on different substrates, such as blood-contacting materials and implanting materials.39, 47 However, Some earlier reports revealed that AgNPs would cause serious hemolysis and cytotoxity when the



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concentrations surpassed a threshold.48-49 So the hemolysis and cytotoxity of the modified membranes were evaluated.

Figure 9. (A) Hemolysis ratios of human red blood cells after incubated with the membranes for 3 h; (B) Results of the in vitro cytotoxicity: ratio of O.D. of L929 cells cultured for 24 h; (C) microscope images of L929 cells cultured for 24 h in extract substrates of the modified membranes.

As shown in Figure 9(A), the membranes with or without AgNPs showed very low hemolysis ratios of about 1 %, indicating that the AgNPs loading did not surpass the threshold, and the coating could be applied on blood-contacting materials. As for cytotoxity, as shown in Figure 9(B), the cell relative growth rate (RGR) for the PES-90-10 and PES-85-15 was comparative with control sample and pure PES membrane (around 99%), indicating that the modified membrane showed no cytotoxicity when the AgNPs loading was below 9.0 µg/cm2. For the PES-80-20 and PES-75-25, the RGR slightly decreased to around 90%, but still above the threshold of cytotoxity. Additionally, most L929 cells incubated in the extract substrates of the modified membranes showed healthy growth morphologies, as shown in Figure 9(C). The results indicated that the designed hydrogel coating showed a cytotoxicity of zero level by precisely controlling the loading amounts of AgNPs, and could be applied on implanting materials. 3.5 Thermo-responsive cell detachment property Currently, in vitro cell culture is one of the most prevalent ways to obtain various species of cells for scientific researches. For in vitro cell culturing, bacteria infections and harvesting of cells are two key issues. Conventionally, antibacterial agent has


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been beforehand added into the cell culture medium to avoid bacterial infections, and proteolytic enzymes (dispase or trypsin) have been used to harvest the cells.50 However, bacteria infections still usually lead to the failure of cell culture, and the enzyme treatment was invasive, which may affect the proper function of the cells.51 In some cases, such as cell sheets engineering, a complete cell sheet is even needed. In order to harvest cells and cell sheets without compromising their functions, thermo-responsive, pH-responsive, and electicity-induced materials have been studied.50, 52 The above analysis has demonstrated that the designed hydrogel coating possessed robust bactericidal property, as well as thermo-responsive bacteria detachment property, but showed no cytotoxity. Therefore, it was anticipated to prepare antibacterial cell culture surface with the property of thermo-responsive detaching cells and cell sheets.53-54 So thermo-responsive cell detachment property was investigated. The thermo-responsive cell detachment property of the hydrogel coating was evaluated by CCK-8 assay, as shown in Figure 10(A). The samples after seeding cells for 48 h were cultured for 30 min at 37 and 4 °C, respectively; then the remained cell numbers were evaluated by CCK-8 assay after rinsing by PBS solution. As shown in Figure 10(B), for the control sample (PS dishes) and pure PES membrane, no significant difference in cell numbers was observed at the temperatures of 37 and 4 °C; while only 20% of the cells were remained for the PES-80-20 at 4 °C compared to that at 37 °C. The cell morphologies were also observed by SEM, as shown in Figure 10(C). The cells spread well on the PES and PES-80-20 membranes after seeding for 48 h. After culturing at 4 °C for 30 min, the cells still spread well with high density on the PES membrane, but only few round cells were observed on the PES-80-20 membrane. Though the cell activity might be limited at 4 °C, the results also indicated that the designed hydrogel coating could detach cells only by changing temperature without enzyme treatments by comparing the results of the hydrogel layer and the pure PES membrane.



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Figure 10. (A) The schematic diagram of thermo-responsive cell detachment property; (B) cell detachment assay tested by CCK-8 method: the samples after seeded cells for 48 h were respectively cultured at 37 and 4 °C for 30 min, then CCK-8 were added to evaluate cell numbers; (C) the cell morphologies on PES and PES-80-20 membranes cultured at 37 and 4 °C, respectively.


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Conclusion Substrate independent AgNPs loaded thermo-responsive hydrogel coating was synthesized and covalently attached onto a model substrate. The porous surface morphologies and chemical compositions of the coatings were characterized by SEM and ATR-FTIR. XRD and EDS-mapping confirmed that AgNPs with a mean diameter of around 108 nm were uniformly distributed into the coating. Above the LCST of the network, the coating showed a hydrophobic surface with low swelling degree, while upon reducing the temperature below LCST, the surface turned to be hydrophilic with high swelling degree. The AgNPs loading amounts could be precisely controlled by regulating the proportions of AA in the hydrogel coatings and the concentrations of Ag+ ions. Meanwhile, the long-term regenerable robust biocidal efficacy could be provided by reloading AgNPs. The contacting and surrounding bacteria could be effectively killed, and the attached dead bacteria could be detached by the improved surface hydrophilicity and swelling degree via reducing the temperature below LCST. The coating also showed good hemocompatibility and cytocompatibility, as well as thermo-responsive cell detachment property. In general, the designed coating showed great potential to be applied to modify the surfaces of various medical devices.

ASSOCIATED CONTENT Supporting Information The detailed descriptions of experiment and characterization methods, and the hydrogel layer thickness and stability analysis were provided in the Supporting Information. This material is free of charge via the internet.

Notes The authors declare no competing financial interest.

Acknowledgements This work was financially sponsored by the National Natural Science Foundation of China (Nos. 51503125, and 51673125), and the State Key Research Development Programme of China (2016YFC1103000). We thank our laboratory members for their generous help.



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Scheme 1. The process of chemically attaching hydrogel thin layer onto PES membrane. 84x51mm (300 x 300 DPI)


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Figure 1. Surface morphologies of the pure and hydrogel layers attached membranes. 145x73mm (150 x 150 DPI)



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Figure 2. (A) ATR-FTIR spectrum of the pure PES membrane and modified PES membrane; (B) XRD spectrum of the AgNPs loaded hydrogel layers. 140x54mm (300 x 300 DPI)


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Figure 3. The mean diameter of AgNPs. 75x27mm (150 x 150 DPI)



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Figure 4. AgNPs distribution in the hydrogel layers analyzed by EDS-mapping. 29x6mm (300 x 300 DPI)


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Figure 5. (A) The water contact angles of the membrnes at 4 and 37 °C; (B) the swelling degree of the attached hydrogel layers at 4 and 37 °C. 140x49mm (300 x 300 DPI)



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Figure 6. (A) AgNPs loading amounts for the modified membranes after each cycle reloading; (B) the releasing profile of AgNPs vs time; (C) the optical degrees of AgNPs loaded membranes for E. coli and S. aureus; (D) the optical degrees of PES-85-15 after different releasing time and several releasing-reloading cycles. 145x107mm (150 x 150 DPI)


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Figure 7. The inhibition zone images of the modified membranes towards E. coli and S. aureus. 75x72mm (150 x 150 DPI)



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Figure 8. The fluorescence images of the bacteria cocultured membranes after immersed and rinsed at 37 °C and 4 °C, respectively. 146x193mm (150 x 150 DPI)


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Figure 9. (A) Hemolysis ratios of human red blood cells after incubated with the membranes for 3 h; (B) Results of the in vitro cytotoxicity: ratio of O.D. of L929 cells cultured for 24 h; (C) microscope images of L929 cells cultured for 24 h in extract substrates of the modified membranes. 124x106mm (300 x 300 DPI)



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Figure 10. (A) The schematic diagram of thermo-responsive cell detachment property; (B) cell detachment assay tested by CCK-8 method: the samples after seeded cells for 48 h were respectively cultured at 37 and 4 °C for 30 min, then CCK-8 were added to evaluate cell numbers; (C) the cell morphologies on PES and PES-80-20 membranes cultured at 37 and 4 °C, respectively. 140x117mm (300 x 300 DPI)


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Substrate-Independent Ag-Nanoparticle-Loaded Hydrogel Coating

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