Thermoresponsive Amphiphilic Functionalization of Thermally

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Biological and Environmental Phenomena at the Interface

Thermoresponsive Amphiphilic Functionalization of Thermally Reduced Graphene Oxide to Study Graphene/Bacteria Hydrophobic Interactions Kok Hui Tan, Shabnam Sattari, Siamak Beyranvand, Abbas Faghani, Kai Ludwig, Karin Schwibbert, Christoph Böttcher, Rainer Haag, and Mohsen Adeli Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b03660 • Publication Date (Web): 06 Mar 2019 Downloaded from http://pubs.acs.org on March 7, 2019

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Thermoresponsive Amphiphilic Functionalization of

2

Thermally Reduced Graphene Oxide to Study

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Graphene/Bacteria Hydrophobic Interactions

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Kok H. Tan,a Shabnam Sattari,b Siamak Beyranvand,b Abbas Faghani, a Kai Ludwig,c Karin

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Schwibbert, d Christoph Böttcher, c Rainer Haag,* a Mohsen Adeli,* a ,b

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a Institut

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Germany

für Chemie und Biochemie, Freie Universität Berlin, Takustr. 3, 14195, Berlin,

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b Department

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c Forschungszentrum

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Chemie und Biochemie, Freie Universität Berlin, Fabeckstr. 36a, 14195 Berlin, Germany

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d Department

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Organisms of Bundesanstalt für Materialforschung und -prüfung, Unter den Eichen 87,

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12205 Berlin, Germany

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nano-interface.

of Chemistry, Faculty of Science, Lorestan University, Khorram Abad, Iran für Elektronenmikroskopie and Core Facility BioSupraMol, Institut für

of Materials and the Environment, Division of Biodeterioration and Reference

KEYWORDS: Graphene, pathogen interactions, hydrophobic interactions, thermoresponsive, 2D

20 21 22

ABSTRACT: An understanding of the interactions of 2D nanomaterials with pathogens is of vital

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importance to develop and control their antimicrobial properties. In this work, the interaction of

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functionalized

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Polyethyleneglycol-block-(poly-N-isopropylacryl-amide) copolymer (PEG-b-PNIPAM) with the

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triazine joint point was attached to the graphene surface by nitrene [2+1] cycloaddition reaction.

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By thermal switching between hydrophobic and hydrophilic states functionalized graphene sheets

graphene

with

tunable

hydrophobicity

and

bacteria

is

investigated.


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were able to bind to bacteria. Bacteria were eventually disrupted, when the functionality was

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switched to the hydrophobic state. On the basis of measuring the different microscopy methods

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and a live/dead viability assay, it was found that Escherichia coli (E. coli) bacteria is more

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susceptible to hydrophobic interactions than B. cereus bacteria, in the same conditions. Our

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investigations confirm that hydrophobic interaction is one of the main driving forces at the

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presented graphene/bacteria interfaces and promotes antibacterial activity of graphene derivatives

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significantly.

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INTRODUCTION

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In the last decade, 2D materials and particularly graphene derivatives have been at the

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forefront of almost every developing field of basic and applied sciences.1 Due to their

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extensive conjugated π-systems, graphene derivatives show unique optical and

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physicochemical properties rendering them suitable for a wide range of applications

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ranging from biosensors to electronic devices.2 Owing to such properties, they have also

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emerged as an attractive candidate for different biomedical applications.3 To produce a high

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performance graphene-based 2D nanomaterial for biomedical applications, e.g.,

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antimicrobial activity, interactions between graphene and biological systems must be

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understood.4 Unfunctionalized graphene is, however, poorly dispersible in aqueous

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solutions and has a strong tendency to agglomerate and to restack. The resulting

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polydispersity, especially with regard to the varying number of stacked layers, makes it

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difficult to control the interaction with biosystems.5 One of the most useful ways to

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overcome such problems is a surface modification of graphene by (macro) molecular

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functional groups.6 This process not only decreases the polydispersity of graphene

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derivatives5 but also offers the possibility to introduce ligands for multivalent interactions

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with biosystems.7 Control over the density of functional groups is a challenging issue,8 but

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simplify studies on the mechanism of these interactions.5, 7c, 8a, 9

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Among the various biosystems, bacteria are of particular interest and they have already been

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investigated in their response towards graphene derivatives.4a,

10

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unfunctionalized graphene sheets have shown efficient antibacterial activity, due to their unique

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mechanical and physicochemical properties.11 Although graphene/bacteria interactions have been

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subjected to extensive scientific studies,11-12 there are still many open questions about graphene

Functionalized and


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antibacterial activity.13 One of the proposed mechanisms for the antibacterial activity of this 2D

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nanomaterial is based on disintegration of the bacteria’s membrane by hydrophobic interactions.14

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However, this mode of action of graphene derivatives has not been studied in more detail as the

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difficult step is to produce graphene sheets with defined functionality. Different functionalized

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graphene derivatives including PEGylated sheets have emerged as promising materials to prevent

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microbe infections, but the molecular mechanisms of bacteria/ graphene interactions is not well

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described.11a,

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understanding the interactions between 2D nanomaterials and bacterial and could be used for the

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construction of smart coatings and surfaces with the ability of trap and release bacteria.

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Switching between hydrophobic and hydrophilic states is a new strategy for

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Recently, we implemented a new method for the controlled nondestructive

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functionalization of carbon based nanomaterials through nitrene [2+1] cycloaddition reaction at

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ambient conditions.6f, 7c, 16 Inspired by these works and using stepwise nucleophilic substitution of

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chlorine atoms of cyanuric chloride, graphene sheets have been developed with a defined

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functionality. Polyethylene glycol (PEG) and poly-N-isopropylacrylamide (PNIPAM) have been

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conjugated to the surface of graphene as a kind of diblock copolymer with the triazine joint point.

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While functionalized graphene sheets remain in solution due to the hydrophilic PEG block, they

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show a thermo-17 and photo-switchable18 hydrophobicity and therefore modified amphiphilicity

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originating from PNIPAM. The synthesized thermosensitive graphene derivatives were then used

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for the investigation of hydrophobic interactions between graphene sheets and bacteria.

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Hydrophobic interactions at graphene/bacteria were investigated by switching between the

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hydrophilic and hydrophobic states. In the present study and for the investigated bacteria,

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hydrophobic interaction is a driving force at the graphene/bacteria interface and has resulted in the

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destruction of bacteria. Different microscopy methods and a live/dead viability assay showed that

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E. coli bacteria are more sensitive to hydrophobic interactions than B. cereus bacteria since their

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membranes were substantially damaged by such interactions.

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EXPERIMENTAL SECTION

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Materials

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Methoxy poly (ethylene glycol) (mPEG) 2000, 2-bromopropionyl bromide (Bpb) (97%), Cu(I)Br

88

(CuBr) (97%), 1,1,4,7,10,10-hexamethyltriethylene tetramine (HMTETA) (97%), fluorescein

89

isothiocyanate isomer I (FITC) (≥ 90%), and N-isopropylacrylamide (NIPAM) (97%) were


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purchased from Sigma Aldrich. Thermally reduced graphene oxide (TRGO) was supplied by Prof.

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Dr. Rolf Mülhaupt from University of Freiburg. Cyanuric chloride (99%) (Trz), sodium azide

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(NaN3) (99%), triethyl amine (TEA) (99%), 4-dimethylaminopyridine (99%) were purchased from

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Acros Organics. Sodium hydroxide (NaOH) (99%) was purchased from Fisher chemical, and

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ethanolamine (Ea) (99%) was purchased from Merck. Biotech Cellulose Ester dialysis bag MWCO

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1000 g mol-1 was purchased from Spectrum labs. E.coli strain (ORN178) OD600~1.0 (1x108 CFU

96

mL-1) was supplied by Dr. Karin Schwibbert and Prof. Dr. Anna Gorbushina from Department of

97

Materials and Environment, Bundesanstalt für Materialforschung und –prüfung and B. cereus

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strain (PTCC 1556 )OD600~1.0 (1x108 CFU mL-1) was obtained from the Pasteur Institute,

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Tehran-Iran. All chemical compounds were used without further purification.

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Synthesis of Triazine-Functionalized Polyethylene Glycol (PEG-Trz)

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PEG-Trz was synthesized according to reported procedure in literature.19 A solution of

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methoxy poly(ethylene glycol) (5 g, 2.5 mmol) and sodium hydroxide (0.2 g, 5 mmol) in

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16 mL distilled water was added dropwise to a solution of cyanuric chloride (4.61 g, 25

105

mmol) in 100 mL dichloromethane at 0 °C. The mixture was stirred at 0/25/50 °C for 1 h,

106

1 h, and 12 hours, respectively. The mixture was then cooled and filtered off and solvent

107

was evaporated to approximately 10 mL. The mixture was then precipitated in diethyl ether

108

at 0 °C and washed with the same solvent for several times. The purified product (5.4 g,

109

2.5 mmol) was obtained as a white solid compound (yield 65%). FTIR: 2907.16 (𝝊𝐂 ― 𝐇),

110

1539.88 (νTCT), 1504.20 (νTCT), 1458.89 (νCH), 1333.53 (νCH), 1099.23 (νCOC) cm-1.

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1H-NMR

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O–triazine), 3.80–3.44 (PEG), 3.39–3.36 (CH3) ppm. Elemental analysis: C: 54.84%, H:

113

8.90%, N: 1.68%, S: 0.00%.

(700 MHz, CDCl3): δ = 4.68 – 4.60 (CH2CH2–O–triazine), 3.88–3.83 (CH2CH2–

114 115

Conjugation of Ethanolamine to PEG-Trz (PEG-Trz-Ea)

116

An excess of ethanolamine (0.84 mL, 14 mmol) was added dropwise into a solution of

117

PEG-Trz (3 g, 1.4 mmol) in dichloromethane (50 mL) at 0 °C. The ice bath was removed

118

then the mixture was stirred at room temperature for 10 hours. Then mixture was

119

concentrated to an approximately 1/3 of the initial volume and it was dialyzed using 1 kD

120

cellulose membrane tube against distilled water for 2 days. The dialyzed mixture was then


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lyophilized at vacuum overnight. The purified product (3.5 g, 1.4 mmol) was obtained as a

122

white solid (yield 95%). FTIR: 3377.25 (νOH, νNH), 2878.24 (νCH), 1567.84 (νTCT),

123

1455.99 (νCH), 1334.50 (νCH), 1092.48 (νCOC) cm-1. 1H-NMR (700 MHz, CDCl3): δ =

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7.90–7.55 (triazine–NH–CH2), 6.55–6.15 (CH2CH2O–triazine), 4.55–4.40 (CH2CH2O–

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triazine), 3.95–3.46 (PEG), 3.41–3.36 (CH3), 3.20–3.12 (OH) ppm. Elemental analysis: C:

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53.13%, H: 8.92%, N: 2.22%, S: 0.00%.

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Conjugation of Azide to PEG-Trz-Ea (PEG-Trz-Ea(N3)

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Excess of sodium azide (0.6 g, 9.2 mmol) was added to a solution of PEG-Trz-Ea (2 g, 0.92

130

mmol) in 30 mL distilled water. The mixture was stirred for 30 minutes and then refluxed

131

at 70 °C overnight. Mixture was cooled down, filtered off, and dialyzed against water using

132

1 kD cellulose ester membrane tube for 2 days. The dialyzed mixture was then lyophilized

133

under vacuum overnight. The purified product was obtained as a white solid (1.7 g, 0.78

134

mmol, 84%). FTIR: 3444.24 (νOH, νNH), 2869.56 (νCH), 2151.20 (νN=N=N), 1456.96 (νCH),

135

1333.53 (νCH), 1094.41 (νCOC) cm-1. Elemental analysis: C: 52.89%, H: 8.50%, N: 3.15%,

136

S: 0.00%.

137 138

Conjugation of PEG-Trz-Ea(N3) onto The Surface of TRGO (PEG-G)

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TRGO was functionalized by [2+1] cycloaddition reactions based on the reported methods

140

in literature.13 TRGO (200 mg) was dispersed in 30 mL dimethylformamide and sonicated

141

for 30 minutes. PEG-Trz-Ea-N3 (1 g, 0.46 mmol) was dissolved in dimethylformamide (5

142

ml) and then added to TRGO dispersion. The mixture was refluxed at 100 °C for 2 days.

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Then it was cooled down and dispersed in acetone and distilled water and centrifuged three

144

times. Product was lyophilized under vacuum for 2 days. FTIR: 2868.59 (𝜐C ― H), 1559.16

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(νTCT), 1455.03 (νCH), 1331.61 (νCH), 1098.26 (νCOC) cm-1. 1H-NMR (700 MHz, CDCl3): δ

146

= 4.00–3.45 (PEG), 3.40–3.30 (CH3) ppm. Elemental analysis: C: 53.55%, H: 8.43%, N:

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2.41%, S: 0.00%.

148 149

Synthesis of Graphene Having Initiator for ATRP Polymerization (PEG-G-Bpb)

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PEG-G (100 mg) was dispersed in dimethylformamide (20 mL) and sonicated for 30

151

minutes. Triethylamine (1.3 μL, 9.2×10-3 mmol) and 4-dimethylaminopyridine (1.1 μg,


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9.2×10-3 mmol) were added to this dispersion and stirred for 1 h at room temperature. 2-

153

Bromopropionyl bromide (9.6 μL, 9.2×10-2 mmol) was added to the mixture dropwise and

154

it was stirred for 2 days at room temperature. Afterwards, product was washed by acetone

155

and water and collected by centrifugation for three times. The final compound was

156

lyophilized under vacuum for 2 days FTIR: 2871.49 (νCH), 1726.94 (νC=O), 1560.13 (νTCT),

157

1455.03 (νCH), 1334.50 (νCH), 1097.29 (νCOC)cm-1.

158 159

ATRP Polymerization of NIPAM Monomer on PEG-G-Bpb

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PEG-G-Bpb (50 mg) was dispersed in distilled water (10 mL) and sonicated for 30 minutes.

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Then, NIPAM (0.7 g, 6.3 mmol) was dissolved in distilled water and added to the above

162

dispersion. The mixture was degassed by freeze-vacuum-thaw procedure and sealed with

163

rubber cap. Cu(I)Br (20 mg, 0.14 mmol) was dissolved in 0.5 mL of above mixture and

164

then injected back into the reaction mixture. HMTETA (40μL, 0.14 mmol) was added in

165

reaction flask dropwise. Mixture was stirred at room temperature for 2 days. Afterwards, it

166

was then centrifuged at 11,000 g for 30 minutes and redispersed in distilled water and the

167

process was repeated three times. The final compound was lyophilized under vacuum for

168

2 days. FTIR: 3276.47 (νNH), 2957.30 (νCH), 2912.95 (νCH), 2859.92 (νCH), 1633.41

169

(νNH), 1521.56 (νTCT), 1448.28 (νCH), 1359.57 (νCH), 1159.97 (νCOC) cm-1. 1H-NMR

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(700 MHz, D2O): δ = 4.10–3.75 (NHCH–(CH3)2), 3.71–3.66 (PEG), 3.50–3.35 (CH3),

171

2.25–1.85 (–CH2–CH–), 1.85–1.35 (–CH2–CH–), 1.30–0.80 (NHCH–(CH3)2) ppm.

172 173

Elemental analysis: C: 60.29%, H: 9.44%, N: 11.11%, S: 0.00%.

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Material characterization

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Nuclear Magnetic Resonance (NMR) spectra were recorded on Bruker ECX 400 MHz, Jeol

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Eclipse 500 MHz, or Bruker AVANCE III 700 MHz NMR spectrometers.

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Fourier Transform Infrared (FTIR) spectra recorded on a Nicolet Avatar 320 FT-IR spectrometer.

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Elemental Analysis was carried out on a VARIO EL III instrument (Elementar, Hanau, Germany)

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using sulfanilic acid as the standard.

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Thermogravimetric Analysis (TGA) was recorded on STA PT 1600 Linseis (Robbinsville, USA)

181

and evaluated with Linseis Data Acquisition software. The measurements were performed in


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aluminum oxide crucibles at a temperatures ranging from 25 °C to 800 °C with a heating rate of

183

10 °C/min.

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Raman Spectra of TRGO derivatives were recorded on the Horiba Jobin-Yvon T64000 with

185

Coherent MBR 110th. The laser was tuned between 650-750 nm for better resolution of the

186

spectra and the intensity was 1000 mW cm-2. Each measurement was taken in a period of

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120 seconds.

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Dynamic Light Scattering (DLS) experiments were performed on Malvern Zetasizer Nano

189

machine (Brookhaven Instruments Corp.) at 25 °C in PBS as solvent. General purpose

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method (NNLS) was used for correlation function to get the distribution of solute’s

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diffusion coefficients (D). The value of hydrodynamic diameter was determined using

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Stokes-Einstein equation. Three repetitive runs with 10 measurements for each run were

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performed to obtain the mean diameter values.

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Photothermal properties of material was measured using Near Infra-red (NIR) irradiation

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with wavelength λ= 785 nm, 500 mW cm-2.

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Bacteria strains were stored at –70℃ in Luria Bertani (LB) medium before use. LB medium was

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prepared by 10 g L–1 peptone, 10 g L– 1 NaCl, then sterilize by autoclaving for 20 min at 15 psi

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(1.05 kg/cm2) on liquid cycle. Bacterium were cultured in Luria-Bertani (LB) medium (5 mL) at

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37 °C for 12 h with the relative humidity less than 90%. The cells culture were washed by

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centrifuged three times with PBS solution at 5000 g; 5 minutes. Bacterial cell suspension was

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diluted in PBS solution to obtain cell samples containing 108 CFU/mL (OD600 ~ 1.0).

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The MIC values were quantified by a microdilution method. In this research, different

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concentrations of samples were prepared in PBS and 100 μL of these solutions were serially diluted

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in a 96-well microplate. Then, 100 μL of freshly prepared microorganism (108 CFU mL-1) was

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added to each well of 96-well microplate and incubated for 24 h at 37 °C. The lowest concentration

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of samples that inhibited the growth of bacteria was defined as the minimum inhibitory

207

concentration.

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In order to image by confocal laser scanning microscopy (CLSM), 100 μL of a FITC solution (1

209

mg mL-1) was added to the 2 mL suspension of bacteria (108 CFU mL-1) in PBS. The mixture was

210

incubated at 4 °C for 2 hours and washed three times with PBS followed the earlier method.

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Prepared sample with different concentrations, was incubated with the FITC labelled bacteria


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(FITC-E. coli and FITC- B. cereus) at 25 and 40 °C and imaged by confocal laser scanning

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microscopy (CLSM).

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Controlled experiments were conducted in parallel without sample on FITC labelled bacteria (E.

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coli and B. cereus) subjected to temperature changes from 25 °C to 40 °C. The fluorescence images

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were recorded on a confocal laser scanning microscope. Morphological studies were made with

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bare bacteria and microscopic images were taken with scanning electron microscope.

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Bacteria Live and Dead Assay by CLSM. 50 μL of graphene derivative suspension (1 mg

219

mL-1, PBS) were added into 500 μL bacteria suspension and incubated at 25 °C or 40 °C

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for 0.5-24 hours. 3 μL of each Syto 9 and propidium iodide fluorescent dye were added

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into the mixture and incubated in the dark place for 15 minutes. Finally, the resulting

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staining was characterized using CLSM microscope. The quantitative result was

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determined from the images in live/dead fluorescent staining assay using imageJ Software.

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The results obtained are mean value from two experiments’ repetition with each experiment

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two to three images were taken into analysis.

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Confocal Laser Scanning Microscopy (CLSM) Experiments. Interactions between

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fluorescence stained E. coli and incubated graphene derivatives compounds were

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monitored on a confocal microscope LSM Leica SP8 machine. 300 μL of the incubated (for

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60 min) compound of concentration 0.09 mg mL-1 was deposited into each well of an 8

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Well ibiTreat μ-Slide (ibidi GmbH, Martinsried, Germany). Then the images were recorded

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by a Leica (DMI6000CSB stand) confocal laser scanning microscope at 63x magnification

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using argon laser (fluorescein isothiocyanate isomer I: excitation 492 nm, emission 500 nm

233

up to 550 nm) and processed by Leica LAS S software. In the case of imaging experiment

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for live and dead assay. Syto 9: excitation 480, emission 490-520 nm; Propidium iodide:

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excitation 530 nm, emission 600-650 nm were applied.

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Cryo-Transmission Electron Microscopy (Cryo-TEM). Perforated carbon film-covered

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microscopical 200 mesh grids (R1/4 batch of Quantifoil, MicroTools GmbH, Jena,

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Germany) were cleaned with chloroform and hydrophilized by 60 s glow discharging at 8

239

W in a BAL-TEC MED 020 device (Leica Microsystems, Wetzlar, Germany) before 5 µl

240

aliquots of the FPS solution (2 mg ml-1) were applied to the grids. Solution of PEG-G-

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PNIPAM in PBS was incubated at least for 30 min at the corresponding temperature. Then

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5 µl of the sample was vitrified by automatic blotting and plunge freezing with a FEI


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Vitrobot Mark IV (Thermo Fisher Scientific Inc., Waltham, Massachusetts, USA)

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operating at the desired temperature and at 100% air humidity and using liquid ethane as

245

cryogen. The vitrified samples were subsequently transferred under liquid nitrogen into a

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Tecnai F20 TEM (FEI Company, Oregon) operating at 160 kV by the use of a Gatan

247

tomography cryo-holder (Model 914) or to the autoloader of a TALOS ARCTICA TEM

248

(Thermo Fisher Scientific Inc., Waltham, Massachusetts, USA) operating at an acceleration

249

voltage of 200 kV. Microscopy was carried out at a 94 K sample temperature using the

250

low-dose protocol of the microscopes. Micrographs were taken with an FEI Eagle 4k × 4k

251

CCD camera using the twofold binning mode (on Tecnai F20) or with a FEI Falcon 3 direct

252

electron detector (on Talos Arctica).

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Scanning Electron Microscopy. A field emission SEM (FE-SEM; TESCAN Company) was used

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to observe morphological changes in bacteria interacting with the PEG-G-PNIPAM. For this

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purpose, the concentration of the E. coli and B. cereus bacteria, were adjusted to 106 CFU/mL after

256

washing with PBS solution. Afterwards, 100 µl of these solutions were incubated with determined

257

amount of the samples at 25 °C and 40° C for 3 hours and 24 hours. The filters were then fixed

258

with 2.5% glutaraldehyde solution for 2 h. Then samples were dehydrated by 10 min incubations

259

in 50, 70, 85, 90, and 100% ethanol. The filters were coated with thin layer of gold.

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RESULTS AND DISCUSSION

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Triazine-functionalized polyethylene glycol (PEG-Trz) was synthesized and changed to a

263

telechelic macromolecule having azide and hydroxyl functional groups by stepwise

264

nucleophilic substitutions (Scheme 1).


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O

O

OH

44

Trz, DCM/H2O NaOH, 50C, 12 h

O

O

44

O

N N

Cl TEA, DCM r.t., 10 h N

O

O

O

44

N

Cl PEG O

O

O

44

H N

N N

N

OH TRGO, DMF 100C, 2 d

OH

N

Cl PEG-Trz-Ea

PEG-Trz

NaN3, H2O 70C, 12 h

H N

N

O

O

44

O

H N

N N

N3

N

Bpb, Et3N, DMAP OH DMF, r.t., 2 d

N

PEG-Trz-Ea(N3) PEG-G HN O

O

44

O

H N

N N

N

O O

Br

N

O NIPAM, HMTETA CuBr, H2O, r.t., 2 d

O 44

PEG-G-Bpb

O

H N

N N

N N

O

Br

O

O

n

HN

O

PEG-G-PNIPAM

265 266

Scheme 1. Synthesis of telechelic polymer and its conjugation to the surface of graphene

267

by nitrene [2+1] cycloaddition reaction. Addition of initiator segments to the functionalized

268

graphene sheets and ATRP polymerization of N-isopropylacrylamide by obtained

269

macroinitiator resulted in PEG-G-PNIPAM.

270 271

Conjugation of this macromolecule to the surface of graphene by nitrene [2+1]

272

cycloaddition reaction and subsequent reaction with α-bromoisobutyryl bromide resulted

273

in the 2D nanomaterial with the ability of initiating atom-transfer radical-polymerization

274

(ATRP) of acrylic monomers.

275

N-isopropylacrylamide (NIPAM) was polymerized on the surface of graphene sheets,

276

and a 2D nanomaterial consisting of graphene, polyethylene glycol (PEG), and poly(N-

277

isopropylacrylamide) (PNIPAM) with the same joint point (PEG-G-PNIPAM) was

278

obtained (Scheme 1). The synthesized materials were characterized by different

279

spectroscopy and microscopy methods as well as thermal and elemental analysis. Figure 1a

280

depicts the IR spectra of the PEG derivatives and functionalized graphene sheets. PEG

281

showed typical absorbance bands at 2874 cm-1 and 1123 cm-1, which corresponded to the


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C-H and C-O-C stretching vibrations. After conjugation of cyanuric chloride to PEG, new

283

absorbance bands could be seen at 1539 cm-1 and 1504 cm-1, which were assigned to the

284

C=N bonds of the triazine ring. One of the chlorine atoms of PEG-Trz was then substituted

285

by ethanolamine. An absorbance band at 3358 cm-1, which corresponded to the hydroxyl

286

functional group of ethanolamine proved the successful synthesis of the PEG-Trz-Ea

287

(Scheme 1). Substitution of the chlorine atom of PEG-Trz-Ea with azide group, resulted in

288

PEG-Trz-Ea(N3). New absorbance band in the IR spectra of this compound can be detected

289

at 2155 cm-1, which is assigned to the azide-stretching vibrations.

290

The PEG derivative was then conjugated onto the surface of TRGO by nitrene [2+1]

291

cycloaddition reaction. Appearance of the absorbance bands of PEG and disappearance of

292

the absorbance band of azide group in the IR spectra of PEG-G confirmed that PEG

293

derivative was conjugated to the surface of graphene sheets by nitrene [2+1] cycloaddition

294

reaction. The carbonyl absorbance band at 1726 cm-1 could be observed in the IR spectra

295

of PEG-G-Bpb, which proved conjugation of the initiator segment to the functionalized

296

graphene sheets. After polymerization of NIPAM monomer by PEG-G-Bpb, several new

297

absorbance bands were observed at 3284 cm-1, 2957 cm-1, 2911 cm-1, and 1635 cm-1, which

298

corresponded to the N-H, CH3, CH2, and carbonyl groups of PNIPAM chains.

299


11

Page 13 of 28

A

B a b c d e f g

NH O

O

O 44

H N

N N

O

HN

e d b

c

Br

e

n O

a

3000 2000 -1 1000 Wavenumber (cm )

C

TRGO (G) PEG-G PEG-G-PNIPAM

600

1200 1800 2400 3000 -1

Wavenumber (cm )

4.0

3.5

3.0

D

2.5 2.0 ppm

1.5

1114.84

372.85

187.58

184.84 0.36 1.00

c

4000

0

O a b

O

N N

d

Relative mass loss (%)

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

Langmuir

1.0

100 75 50 25 0

TRGO (G) PEG-G PEG-G-PNIPAM

200 400 600 Temperature (°C)

800

300 301

Figure 1. (A) IR spectra: (a) PEG, (b) PEG-Trz, (c) PEG-Trz-Ea, (d) PEG-Trz-Ea(N3) (e)

302

PEG-G, (f) PEG-G-Bpb, and (g) PEG-G-PNIPAM. (B) 1H-NMR spectrum of PEG-G-

303

PNIPAM (500 MHz, D2O). (C) Raman spectra of TRGO, PEG-G, and PEG-G-PNIPAM.

304

D and G bands can be clearly recognized at 1350 cm-1 and 1587 cm-1, respectively. (D)

305

TGA thermograms of TRGO, PEG-G, and PEG-G-PNIPAM.

306 307

Figure 1B shows 1H NMR spectrum of PEG-G-PNIPAM in which different signals

308

of PNIPAM chains as well as a weak signal for PEG can be recognized. Figure 1C shows

309

Raman spectra of TRGO and the functionalized graphene sheets. TRGO shows a high D/G

310

ratio, due to the highly defected structure of this compound. These features can be seen in

311

the Raman spectra of PEG-G and PEG-G-PNIPAM confirming the presence of TRGO in

312

their structure.


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TRGO, PEG-G, and PEG-G-PNIPAM were analyzed by TGA (Figure 1D). TRGO

314

showed a decomposition temperature between 470 °C - 700 °C. After conjugation of

315

polyethylene glycol to the surface of TRGO, two weight loss regimes at 200 - 410 °C and

316

410 - 700 °C were observed corresponding to the loss of PEG and TRGO segments,

317

respectively. According to TGA experiments, the graphene content of PEG-G was less than

318

10%. Thermal behavior of PEG-G-PNIPAM was very similar to PNIPAM, indicating that

319

the major part of this compound is poly (N-isopropyl-acrylamide). According to these

320

analyses, the graphene and PEG contents of PEG-G-PNIPAM are less than a few percent.

321

The nitrogen content of PEG derivatives and functionalized graphene sheets was

322

used as an indicator for characterization of their structures. Comparison of the experimental

323

and calculated nitrogen content (Table S1) revealed that the density of PEG chains attached

324

to the surface of graphene sheets was one chain per 62 graphene carbons (See ESI). After

325

polymerization of NIPAM onto the surface of PEG-G, the nitrogen content significantly

326

increased due to the long PNIPAM chains.


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Page 15 of 28

b c

55

c

a b ab n n HN O HN

O

c d c d

44

d d b a

33 22 ppm ppm

25°C 25°C 30°C 30°C 35°C 35°C 40°C 40°C 11 00

40

Intensity (%)

a

LCST ~34°C

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

Langmuir

30 20

25°C 30°C 35°C 40°C

10 0 100

1000 Size (nm)

327 328

Figure 2. (a) Photograph of a PBS solution (1 mg mL-1) of PEG-G-PNIPAM at

329

temperatures lower and higher than its LCST. (b) 1H NMR spectra (500 MHz, D2O) and

330

(c) DLS diagrams of PEG-G-PNIPAM at 25 °C, 30 °C, 35 °C, and 40 °C. (d) and (e) Cryo-

331

TEM images of PEG-G-PNIPAM at 25 °C and 40 °C. Contour of the graphene assemblies

332

is enhanced by dotted lines. Scale bar is 100 nm.

333 334

Interactions between bacteria and functionalized graphene sheets

335

Since one of the proposed mechanisms for the antibacterial activity of graphene derivatives

336

is based on hydrophobic interactions,14, 20 PEG-G-PNIPAM sheets were used to investigate

337

the validity of this mechanism, because its thermoresponsive behavior could be used for

338

triggering hydrophobicity.

339

Several previous studies have reported possible mechanisms for the antibacterial

340

activity of graphene including ROS generation, “nano-knives” or “sharp edges effect,” and


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Page 16 of 28

341

wrapping or trapping bacteria with GO sheets.6g, 10c, 21 It is known that these mechanisms

342

strongly depend on the direct contact between graphene derivatives and bacteria10a-c, 10e. In

343

order to eliminate these mechanisms and amplify hydrophobic interactions, amphiphilic

344

graphene derivatives with polymeric barrier and defined lower critical solution temperature

345

(LCST) are synthesized. The thick polymeric shell around the graphene sheets by longer

346

PNIPAM

347

mechanisms.10a, 11a, 12c Also the polymeric shell can minimize the changes in the chemical

348

state of graphene sheets that can occur in the cell culture medium or by bacteria

349

respiration.22 The weight ratio of graphene, PEG, and PNIPAM was adjusted to 3%, 10%,

350

and 87%, respectively (see detail in ESI). Consequences of such composition, which are

351

discussed below, confine interactions between graphene sheets and bacteria to hydrophobic

352

interactions.

chains

prevents

antibacterial

activity

through

the

above-mentioned

353

The high content of long PNIPAM chains allow one to induce a photothermal-tunable

354

hydrophobic character, while shorter PEG chains facilitate a permanent water

355

dispersibility.

356

The small amounts of graphene and PEG do not significantly affect the LCST of

357

PNIPAM. PEG-G-PNIPAM with the above-mentioned properties should therefore be

358

suitable to study the specific role of hydrophobic interactions at the graphene/bacteria

359

interface.

360

The cloud point of PEG-G-PNIPAM could be observed at about 34 °C. (Figure 2a). 1H NMR

361

was used to further investigate the LCST of PEG-G-PNIPAM. While signals of PNIPAM chains

362

could be clearly observed up to 30 °C, they diminished at higher temperatures and disappeared

363

completely at 40 °C (Figure 2b). This indicates that the PNIPAM chains are collapsed on the

364

surface of graphene transforming it to a hydrophobic state. It is worth noting that aqueous

365

dispersions of the functionalized graphene sheets were stable at 40 °C for several days. While the

366

graphene surface is hydrophobic at these conditions, its aqueous dispersion is still enabled by the

367

PEG chains.

368

Correlation between morphology of graphene sheets and temperature was investigated by

369

dynamic light scattering (DLS) and cryo-TEM. According to DLS experiments, average size of

370

graphene sheets decreased from ~950 nm to ~430 nm when the solution temperature was increased

371

from 25 °C to 40 °C (Fig. 2c). This indicates that graphene sheets do not form larger agglomerates


15

Page 17 of 28

372

at temperatures higher than LCST. Cryo-TEM was employed to investigate the morphology of

373

PEG-G-PNIPAM in PBS at different temperatures.

374 a Temperature (°C)

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

Langmuir

50

PEG-G-PNIPAM E. coli

b

Temp. of the irradiated spot

g

40 30 20 0

200 400 Time (s)

IR irradiated spot

600

Photothermal experiment

c

d

PEG-G-PNIPAM + E. coli (25°C)

i

E. coli (25°C)

e

375

h

f

E. coli (40°C)

j


k

l


376

Figure 3. (a) and (b) Evaluation of the photothermal conversion of PBS solution of PEG-G-

377

PNIPAM (1 mg mL-1) under NIR laser irradiation (785 nm, 500 mW cm-2). (c) and (e) are CLSM

378

images of FITC-labeled E. coli at 25 °C and 40 °C, respectively. (d) and (f) correspond to bright

379

field images of (c) and (e), respectively. CLSM images of FITC-labeled E. coli incubated with

380

PEG-G-PNIPAM at (g) 25 °C, (i) 40 °C, and (k) subsequently cooled down to 25 °C. (h), (j), and

381

(l) are the corresponding bright field images of (g), (i), and (k), respectively.

382 383

At 25 °C, functionalized graphene sheets were in a flat state (Figure 2d). The flat morphology

384

of graphene sheets is due to the hydrophilic PEG and PNIPAM chains. Raising the solution


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Page 18 of 28

385

temperature to 40 °C and thereby increasing the hydrophobicity of the graphene sheets resulted in

386

a transition from a flat to a rolled up, cylindrical shape (Figure 2e).

387

The photothermal property of PEG-G-PNIPAM was evaluated by laser irradiation

388

(785 nm, 500 mW cm-2) for 10 minutes. As shown in Fig. 3a and 3a', a short irradiation (90

389

seconds) was sufficient to achieve the LCST (34 °C) of the system.

390

To test the hydrophobic interactions between graphene sheets and bacteria, PEG-G-

391

PNIPAM was exposed to Escherichia coli (Gram-negative) and B. cereus (Gram-positive)

392

in aqueous solution.

393

PEG-G-PNIPAM was incubated with the FITC-labeled E. coli (FITC-E. coli) at 25

394

and 40 °C and imaged by confocal laser scanning microscopy (CLSM). Due to the Förster

395

resonance energy transfer (FRET) effect, the interaction between the FITC-E. coli and

396

graphene sheets should have resulted in fluorescence quenching. Accordingly, partial

397

quenching of the fluorescence of E. coli upon incubation with PEG-G-PNIPAM at 25 °C

398

occurred, which confirmed weak interactions between graphene sheets and bacteria at this

399

temperature (Figure 3b, videos 1 and 2). Interestingly, a strong fluorescence quenching was

400

observed at 40 °C (Figure 3d, videos 3 and 4). This result proves that graphene sheets were

401

close enough to the membrane of bacteria to quench its fluorescence by FRET mechanism.

402

Efficient fluorescence quenching occurred, when the distance between graphene surface

403

and probe was less than 4 - 7 nm.23 It is worth noting that the total mass of graphene in

404

PEG-G-PNIPAM accounted for only few percent, but an efficient fluorescence quenching,

405

which is an indicator for far-reaching graphene/bacteria interactions, was achieved. The

406

efficiency of fluorescence quenching is further highlighted, when the rolled up morphology

407

and decreased surface area of graphene sheets at 40 °C is considered. Interactions between

408

PEG-G-PNIPAM and E. coli were reversible, and a regeneration of fluorescence was

409

observed when the temperature was reduced below LCST (Figure 3f, video 5). However,

410

switching between hydrophobic and hydrophilic states resulted in a partial irreversible

411

fluorescence quenching (Figure 4a and b). This could be due to the weak hydrophobic

412

interactions at 25 °C or disruption of bacteria membranes by these interactions.

413

The quenching of fluorescence was more efficient for E. coli than B. cereus. The

414

fluorescence intensity of FITC-E. coli was decreased to 16% after 8 times switching; while

415

it retained at 81% for the FITC-labeled B. cereus after the same number of switching. These


17

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Langmuir

416

results show that the hydrophobic interactions between PEG-G-PNIPAM and E. coli

417

bacteria were stronger than that between PEG-G-PNIPAM and B. cereus bacteria. It has

418

been recently shown that proteins containing large amount of hydrophobic amino acids

419

accelerated hydrophobic interactions between graphene derivatives and bacteria.4a Gram-

420

negative bacteria possess an outer membrane which contains lipopolysaccharides on its

421

outer leaflet but membrane of gram-positive bacteria is surrounded by peptidoglycans.

422

Therefore stronger hydrophobic interactions between Gram-negative bacteria and graphene

423

are due to the composition of its membrane. Lipopolysaccharides on the outer layer of

424

membrane of E. coli increase its hydrophobic interaction with graphene sheets. Such

425

hydrophobic interactions could disrupt the integrity of bacteria’s membrane.

426

In order to investigate the effect of such interactions on bacteria, the viability of E.

427

coli and B. cereus after incubation with graphene sheets was studied using a live/dead

428

bacterial viability assay. While antibacterial activity of PEG-G-PNIPAM against B. cereus

429

at 25 °C was negligible, more efficacy was observed against E. coli under the same

430

conditions (Figure 4 c and d). The minimum inhibitory concentration (MIC) of graphene

431

nanosheets against pathogenic bacteria was quantified by a microdilution method. The

432

highest effect was observed for PEG-G-PNIPAM by switching the temperature to higher

433

than LCST. While MIC of this compound against E .Coli at 25 °C was 1500 μg/ml, it

434

decreased to 1000 μg/ml at 40 °C.

435


18

Langmuir

a

Graphene/E.coli

100

b

E.coli

60 40 20

1

2

3

4

5

6

7

8

9

40 20

150

d

Control 25 °C Control 40 °C Graphene/25 °C Graphene/40 °C

120

0

Cell viability (%)

c

0

10

Time (h)

90 60 30

Bacillus

60

0 0

Graphene/Bacillus

100 80

Fluorescence intensity (ABU)

Fluorescence intensity (ABU)

80

Cell viability (%)

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

Page 20 of 28

1

2

3

4

5

6

7

8

9

10

Time (h) 150

Control 25 °C Control 40 °C Graphene/25 °C Graphene/40 °C

120 90 60 30 0

0 0

3

6

9

12

15

18

21

24

0

3

6

9

12

15

18

21

24

Time (h)

Time (h)

436

Figure 4. Fluorescence quenching of FITC-labeled (a) E. coli and (b) B. cereus after

437

incubation with PEG-G-PNIPAM and consecutive switching to temperatures higher and

438

lower than LCST. Antimicrobial activity of PEG-G-PNIPAM (100 µg/mL) against (c) E.

439

coli and (d) B. cereus at different incubation times and temperatures. The quantitative result

440

was determined from the images in live/dead fluorescent staining assay using image J

441

software (n=3).

442 443 444

This result, together with a minor fluorescence quenching at 25 °C, suggest weak hydrophobic interaction between E. coli and bacteria even at room temperature.

445

However, antibacterial effectivity of the functionalized graphene sheets was

446

substantially enhanced by switching the temperature to 40 °C. In accordance with the

447

fluorescence quenching studies (Figures 4a and b), the lower activity of PEG-G-PNIPAM

448

against B. cereus at 40 °C proved that Gram-positive bacteria were less susceptible to

449

hydrophobic interactions than their Gram-negative analogs.


19

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Langmuir

450 451 452

Figure 5. SEM images of B. cereus incubated with PBS at (a) 25 °C for 3h, (c) 40 °C for

453

3h, (e) 25 °C for 24 h, and (g) 40 °C for 24 h. SEM images of B. cereus incubated with

454

PEG-G-PNIPAM at (b) 25 °C for 3h, (d) 40 °C for 3h, (f) 25 °C for 24 h, and (h) 40 °C for

455

24 h.

456 457

Hydrophobic interactions between graphene sheets and bacteria were further

458

investigated by SEM imaging (Figures 5 and 6). The membrane of B. cereus was affected

459

by the interaction with the functionalized graphene sheets only at 40 °C, while no

460

significant effect could be observed at ambient temperatures (Figure 5). However, the

461

membrane of E. coli was already damaged at ambient temperatures when it was incubated

462

with PEG-G-PNIPAM (Figure 6).


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Page 22 of 28

463 464

Figure 6. SEM images of E. coli incubated with PBS at (a) 25 °C for 3h, (c) 40 °C for 3h,

465

(e) 25 °C for 24 h, and (g) 40 °C for 24 h. SEM images of E. coli incubated with PEG-G-

466

PNIPAM at (b) 25 °C for 3h, (d) 40 °C for 3h, (f) 25 °C for 24 h, and (h) 40 °C for 24 h.

467 468

As mentioned above, this antibacterial activity could be assigned to the weak

469

hydrophobic interactions between graphene and E. coli at ambient conditions. When

470

temperature was increased to 40 °C, the effect was amplified up to the complete disruption

471

of the bacterial membrane after longer incubation times.

472

Differences between antibacterial activity of PEG-G-PNIPAM against E. Coli and B.

473

cereus as well as correlation between this activity and hydrophobicity of graphene sheets,

474

supported by fluorescence quenching, live/dead assays, and SEM images, emphasize the crucial

475

role of hydrophobic interactions at graphene/bacteria interfaces.

476

The ability of graphene sheets to extract lipid from the membrane of bacteria has been

477

reported before but the mechanism is not fully investigated yet.24 Our data show that one of the

478

main driving forces in the bacteria/graphene interfaces is hydrophobic interaction. This is in the

479

same line with other proposed mechanisms and reported antibacterial activity in the literature. The

480

hydrophobic interactions between graphene sheets and bacteria can add a synergic effect to the

481

other driving forces including mechanical15c,

482

antibacterial activity of graphene derivatives. Diversity of the structure and physicochemical

25

and chemical26 stresses and accelerate the


21

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Langmuir

483

properties of graphene derivatives broaden the range of mechanisms or at least the coefficients of

484

each mentioned parameter at graphene/bacteria interface. Therefore, proposed mechanisms for the

485

antibacterial activity of graphene sheets should be correlated with their structures, functionality,

486

and external environmental parameters, including cell culture conditions as well as the bacteria

487

strain and state, such as state of the cell growth and cell density.

488 489

CONCLUSION

490

Functionalized graphene sheets with thermally switchable amphiphilicity were synthesized and

491

used to investigate hydrophobic interactions at graphene/bacteria interface. In the present study,

492

the hydrophobic interactions play a crucial role at graphene/bacteria interface. Large graphene

493

sheets with inaccessible edges slowly penetrate into the membrane of bacteria and their short-term

494

(30 minutes) interactions with membrane of bacteria are reversible but destructive at longer

495

incubation times. Gram-negative bacterium was more susceptible to hydrophobic interactions than

496

Gram-positive bacterium, due to their membrane composition. The thermosensitive functionalized

497

graphene sheets could be applied for the in vivo tests, because they are highly water dispersible

498

and switch to the hydrophobic state at body temperature. Taking advantages of the

499

thermosensitivity of these materials, they can be used for the construction of smart antibacterial

500

coating and related medical devices.

501 502

ASSOCIATED CONTENT

503

Supporting Information

504

The Supporting Information is available free of charge on the ACS Publications website at DOI:

505

xx.xxxx/xxxxxx.xxxxxxx.

506

Characterization and supporting data of the materials (PDF)

507 508

AUTHOR INFORMATION

509

Corresponding Authors

510

*Email: [email protected]

511

*Email: [email protected]

512

ORCID

513

Kok H. Tan: 0000-0002-3975-5270


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514

Rainer Haag: 0000-0003-3840-162X

515

Mohsen Adeli: 0000-0001-6895-8491

Page 24 of 28

516 517

Author Contributions

518

The project was supervised by Rainer Haag and Mohsen Adeli. Synthesis of materials and a part

519

of biological studies were performed by Kok H. Tan and Abbas Faghani. Shabnam Sattari,

520

Siamak Beyranvand, and Karin Schwibbert collaborated in bacterial interactions. Kai Ludwig

521

and Christoph Böttcher performed cryo-TEM studies.

522 523

Notes

524

There are no conflicts to declare.

525 526

ACKNOWLEDGMENT

527

We thank the collaborative research center SFB 765 of the Deutsche Forschungsgemeinschaft for

528

financial support. We would like to acknowledge Dieter Treu for operating the XPS instrument at

529

BAM. We also thank support by the team at the BESSY II synchrotron radiation facility as well

530

as Dr. A. Nefedov (Karlsruhe Institute of Technology, KIT) from the HE-SGM Collaborate

531

Research Group. We acknowledge Dr. Pamela Winchester for language polishing the manuscript.

532 533

REFERENCES

534 535 536 537 538 539 540 541 542 543 544 545 546 547

1. Li, X.; Cai, W.; An, J.; Kim, S.; Nah, J.; Yang, D.; Piner, R.; Velamakanni, A.; Jung, I.; Tutuc, E.; Banerjee, S. K.; Colombo, L.; Ruoff, R. S., Large-Area Synthesis of High-Quality and Uniform Graphene Films on Copper Foils. Science 2009, 324 (5932), 1312-1314. 2. Geim, A. K.; Novoselov, K. S., The rise of graphene. Nat. Mater. 2007, 6 (3), 183-191. 3. (a) Yin, P. T.; Shah, S.; Chhowalla, M.; Lee, K.-B., Design, Synthesis, and Characterization of Graphene–Nanoparticle Hybrid Materials for Bioapplications. Chemical Reviews 2015, 115 (7), 2483-2531; (b) Mao, H. Y.; Laurent, S.; Chen, W.; Akhavan, O.; Imani, M.; Ashkarran, A. A.; Mahmoudi, M., Graphene: Promises, Facts, Opportunities, and Challenges in Nanomedicine. Chemical Reviews 2013, 113 (5), 3407-3424. 4. (a) Romero-Vargas Castrillón, S.; Perreault, F.; de Faria, A. F.; Elimelech, M., Interaction of Graphene Oxide with Bacterial Cell Membranes: Insights from Force Spectroscopy. Environ. Sci. Technol. Lett. 2015, 2 (4), 112-117; (b) Parlak, O.; Turner, A. P. F.; Tiwari, A., On/OffSwitchable Zipper-Like Bioelectronics on a Graphene Interface. Advanced Materials 2014, 26 (3), 482-486.


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Thermoresponsive Amphiphilic Functionalization of Thermally

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