Hydrophobically Modified Chitosan-Grafted Magnetic Nanoparticles

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Hydrophobically Modified Chitosan Grafted Magnetic Nanoparticles for Bacteria Removal Duc-Thang Vo, Chris George Whiteley, and Cheng-Kang Lee Ind. Eng. Chem. Res., Just Accepted Manuscript • DOI: 10.1021/acs.iecr.5b01335 • Publication Date (Web): 14 Sep 2015 Downloaded from http://pubs.acs.org on September 14, 2015

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Industrial & Engineering Chemistry Research

1

Hydrophobically Modified Chitosan Grafted Magnetic Nanoparticles for

2

Bacteria Removal

3

Duc-Thang Voa, Chris G. Whiteleyb, Cheng-Kang Leea,*

4

a

Department of Chemical Engineering, b Graduate Institute of Applied Science &

5

Technology, National Taiwan University of Science and Technology, Taipei 106, Taiwan.

6

ABSTRACT: Hydrophobically modified chitosan (HMCS), synthesized by reacting dodecyl

7

aldehyde with chitosan (CS) has good hemostatic properties and can, by means of its

8

hydrophobic tail, coagulate blood cells. In this work, the ability of synthesized HMCS to

9

coagulate Escherichia coli cells was demonstrated. In order to facilitate the removal of

10

coagulated E. coli cells using an applied magnetic field, HMCS was grafted on to the surface of

11

magnetic nanoparticles (MNPs). Such modified MNPs interacted with Gram-negative bacteria

12

such as E. coli by means of strong hydrophobic forces between the hydrophobic tails of HMCS

13

and outer membrane of E. coli. The highest E. coli removal capacity achieved by MNPs@HMCS

14

was 1.38 x 108 cells/mg. The characterization of CS, HMCS, CS/HMCS grafted MNPs

15

(MNPs@CS, MNPs@HMCS) were carried out by Fourier transform infrared spectroscopy

16

(FTIR), elemental analysis (EA), scanning electron microscopy (SEM) and thermal gravimetric

17

analysis (TGA). The degree of deacetylation (DDA) and degree of substitution (DS) of the

18

synthesized HMCS are 81% and 11%, respectively.

19

Keywords: hydrophobically modified chitosan, magnetic nanoparticles, E. coli removal, E. coli

20

coagulation.

21

INTRODUCTION

22

Chitosan is a well-known cationic biopolymer obtained from chitin after thorough 1 ACS Paragon Plus Environment


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deacetylation. Due to its biocompatibility, antimicrobial activity, wound healing property, and

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many other features, chitosan and its derivatives have made application in the pharmaceutical

25

industry, biomedicine, water treatment, cosmetics, agriculture and food industry

26

many of its derivatives, the hydrophobically modified chitosan (HMCS), prepared by attaching

27

the hydrophobic tails to the amino groups of chitosan has been used as drugs carrier for the

28

treatment of cancer cells. Due to the hydrophobized interaction, HMCS is able to formed self-

29

assembled nanoparticles with hydrophobic core that can entrap and carry many hydrophobic

30

antitumor drugs 4, 5 4 6. It has also been reported that HMCS is easier to be internalized in cancer

31

cells than in normal cells 7. Besides, the cellular uptake of HMCS nanoparticles (N-palmitoyl

32

chitosan) has been demonstrated to be increased with the degree of substitution of hydrophobic

33

tails on chitosan backbone due to the significant increase in the intra- and intermolecular

34

hydrophobic interactions with the cell membranes 8.

1-3

. Among

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Due to its hydrophobic tails, HMCS has a very unique gel formation property. It was reported

36

that HMCS solution turned into a gel when in contact with a suspension of hydrophobic carbon

37

nanospheres 9. The gelation of this HMCS solution was attributed to the strong hydrophobic

38

interaction between hydrophobic tails of HMCS and carbon nanospheres that lead to the

39

formation of a 3-D network. HMCS was also demonstrated to have hemostatic properties by

40

inducing blood cell coagulation

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membrane anchoring it to the polymeric chain of HMCS. The blood cells play the role as a 3-D

42

network of nodes that form a gel. In addition, HMCS was able to convert several mammalian

43

cells suspensions, including human or bovine blood, endothelial cells, and breast cancer cells

44

into reversible gels 11. Consequently, based upon a similar mechanism, the hydrophobic tails of

45

HMCS are expected to be inserted into the lipid bi-layer of the bacterial cell membrane thereby

10

through insertion of its hydrophobic tail into the blood cell

2 ACS Paragon Plus Environment

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inducing the gelation of a bacterial suspension. Magnetic nanoparticles (MNPs) have numerous potential biochemical related applications 12-15

. The

48

owing to their magnetic, nontoxicity, biocompatibility, and biodegradability properties

49

magnetic property makes the nanoparticles easy to handle with an applied external magnetic

50

field. Besides, the surface of the magnetic nanoparticles can be modified with various functional

51

groups for attaching affinity ligands for bio-specific separation and molecular targeting.

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Chitosan has often been used to modify the surface of MNPs by co-precipitation with magnetic

53

iron oxide from the alkaline ferric/ferrous solution because of its acid-soluble but alkali-insoluble

54

property. The chitosan-MNPs complex obtained by this co-precipitation has been used for heavy

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metal ion adsorption, enzyme immobilization, affinity proteins adsorption and many other

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applications

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able to specifically capture the vancomycin-resistant enterococci and Gram-positive bacteria at

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an ultralow concentration

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surface were highly efficient for rapid pathogen detection and decontamination 22.

18, 20

16-19

.

. Recently, magnetic nanoparticles with vancomycin grafted on its surface were

21

. Magnetic nanoparticles with modified sugars grafted on their

60

This present work takes advantage of the strong cell membrane interaction of hydrophobic

61

modified chitosan to modify the surface of commercially available magnetic nanoparticles and to

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capture and remove pathogenic bacteria. The work is divided into three sections: First HMCS

63

was synthesized, characterized and its ability to induce gelation of bacterial solution

64

demonstrated. Second HMCS was covalently grafted to the surface of MNPs via Schiff base

65

reaction to form HMCS modified MNPs (MNPs@HMCS). Third pathogenic E. coli as well as

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Staphylococcus aureus were decontaminated using the synthesized MNPs@HMCS.

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EXPERIMENTAL SECTION

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Materials. Chitosan, from shrimp shells (MM ~ 512 kDa, calculated by intrinsic viscosity 3 ACS Paragon Plus Environment


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using the Mark–Houwink–Sakurada equation) 23 with a degree of deacetylation of approximately

70

72% was purchased from Sigma-Aldrich. Magnetic nanoparticles (Bayoxide E 8706) were

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obtained from Lanxess Energizing Chemistry, Germany. Sodium silicate (Na2SiO3.9H2O),

72

tetraethoxysilane (TEOS, 98%), 3-aminopropyltriethoxy silane (APTES, 99%), sodium

73

cyanoborohydride (NaCNBH3, 95%), ninhydrin (reagent ACS), dodecyl aldehyde (92%), sodium

74

phosphate, monobasic monohydrate (NaH2PO4.H2O, 99%), sodium phosphate, dibasic

75

heptahydrate (Na2HPO4.7H2O, 99%), sodium chloride (NaCl, 99.5%), agar were purchased from

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Acros. Acetic acid glacial (analytical reagent grade), sodium hydroxide (NaOH, Laboratory

77

Reagent Grade), ammonia solution (35%, analytical reagent grade) were purchased from Fisher

78

Scientific. Glutaraldehyde (GA, 25% aqueous solution) was purchased from Alfe Aesar. E. coli

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BL21 (Novagen, Madison, WI, USA) and S. aureus ATCC6538P (Taiwan Textile Research

80

Institute).

81

Hydrophobically modified chitosan (HMCS). Chitosan (4 g), dissolved in acetic acid (220

82

mL, 0.2M) was diluted in ethanol (150 mL) and the pH adjusted to pH 5.1 with NaOH solution

83

(1M) to prevent precipitation of the chitosan. The homogeneous solution was treated with

84

dodecyl aldehyde dissolved in ethanol (10 mL, 0.025 g/mL). An excess amount of sodium

85

cyanoborohydride (3 moles per chitosan monomole) was then added, at room temperature, with

86

stirring for 24 h. The alkylated chitosan was precipitated from solution by adjusting to pH 7

87

using NaOH solution (1M) followed by an excess of ethanol. The precipitate was collected,

88

washed thoroughly with ethanol to remove unreacted dodecyl aldehyde and sodium

89

cyanoborohydride

90

residual sodium cyanoborohydride in the precipitate was checked by ninhydrin assay 25.

91

24

and the product HMCS dried under vacuum at room temperature. Any

CS/HMCS surface grafted magnetic nanoparticles (MNPs@CS, MNPs@HMCS). The 4 ACS Paragon Plus Environment

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.

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surface modification of MNPs for CS or HMCS was based on that reported in the literature

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Briefly MNPs (2 g) was dispersed in 120 mL aqueous solution containing 4.6 g Na2SiO3.9H2O

94

and 0.4 g NaOH. After ultrasonication (5 min, ultrasonic bath Branson 5800), the pH of the

95

MNPs suspension was adjusted to 6 by the slow addition of HCl (2 M). Particles, collected from

96

the solution by a magnet, were washed thoroughly with deionized water and designated as

97

MNPs@SiO2. They were further coated with a porous silica layer by using a sol–gel process

98

based on the hydrolysis of TEOS in ethanol/ammonia solution. The collected MNPs@SiO2 was

99

dispersed by ultrasonication, into a solution that consisted of 50 mL ethanol, 50 mL deionized

100

water and 1 mL concentrated ammonia solution (28 wt%). TEOS solution (0.2 mL TEOS in 10

101

mL of ethanol) was then added at a rate of 1 mL/min under mechanical stirring and the reaction

102

was allowed to proceed at 850C for 0.5 h. Thus MNPs@SiO2@TEOS was obtained after washing

103

several times with deionized water and drying at 600C overnight.

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After MNPs@SiO2@TEOS was well dispersed in 50 mL ethanol, 1 mL APTES was added

105

with mechanical stirring and the reaction was continued at room temperature for 1 h. This

106

surface aminated magnetic nanoparticles (MNPs@APTES) was activated with glutaraldehyde

107

(10 mL, 25 %) in phosphate buffer (50 mL 0.1 M, pH 7.4) and sodium cyanoborohydride (1.0

108

mL, 0.1 mg/mL) at room temperature (6 h).

109

glutaraldehyde activated MNPs (MNPs@GA) via Schiff base reaction. MNPs@GA was stirred

110

(24 h, room temperature) with CS/HMCS (250 mL, 1 mg/mL in 0.2 M acetic acid containing 0.3

111

g NaCNBH3 The obtained CS/HMCS grafted MNPs were washed, collected and dried under

112

vacuum at room temperature until further use. The reaction scheme for the surface modification

113

of MNPs is shown (Scheme 1).

CS or HMCS was then grafted onto the



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Scheme 1. Schematic representation of the preparation of HMCS grafted MNPs (MNPs@HMCS).

117

Characterization. Fourier transform infrared (FTIR) spectra of CS, HMCS and the surface

118

modified MNPs were recorded using a FTS-3500 FTIR spectrophotometer at room temperature

119

using KBr pellet. Steady viscosity of CS/HMCS solution and bacterial cells suspensions mixed

120

with CS/HMCS were measured using a MCR102 Modular Compact Rheometer at room

121

temperature 10. The morphology and size of MNPs, E. coli and E. coli attached MNPs@HMCS

122

were observed by a field emission scanning electron microscope (Joel, model JSM-6500F). To

123

take the SEM images of bacterial cells coagulated by MNPs@HMCS, the suspension of

124

coagulated sample was dropped on a microporous mixed-cellulose ester (MCE) membrane, and

125

rinsed three times with sterile PBS (0.01 M, pH = 7.4), then fixed in 5% (v/v) glutaraldehyde

126

prepared in PBS solution for 30 min at room temperature. The glutaraldehyde fixed samples

127

were immersed in 25%, 75%, 100% ethanol stepwise for dehydration and dried at room

128

temperature 26 27. Thermal gravimetric analysis (TGA) with a heating rate of 100C/min (Diamond


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TG/DTA, PerkinElmer Instrument) was done to measure the amount of chitosan or HMCS

130

grafted onto MNPs. The amount of amino and aldehyde groups on the surface of MNPs were

131

determined by ninhydrin assay and sulfuric acid – phenol assay using glycine and GA as

132

standards, respectively 28. The net electrostatic charge of MNPs, MNPs@CS and MNPs@HMCS

133

(pH 7.4) were characterized by using a zeta potential meter (PALS Zeta Potential Analyzer Ver.

134

5.73).

135

Bacterial cells suspension gelation. Bacterial (E. coli and S. aureus) suspension (1 mL, 50%,

136

w/w, in DI water) was mixed with CS or HMCS solution (0.3%, w/v, in 0.2 M acetic acid) at 5:1,

137

5:2, 5:3 volume ratio, respectively, in a test tube. In order to test the extent of gelation the test

138

tube was inverted and the sample solution claimed to have suitable gelation capability if the

139

mixture was not able to flow within 3 min.

140

Pathogenic cells capture. The capability of MNPs to capture pathogenic bacteria was 29, 30

141

evaluated by using an optical density method as described elsewhere

142

grown on LB nutrient agar were selected using a wire loop and placed in 5 mL of LB medium,

143

which was then incubated at 370C overnight to activate the bacteria. This overnight E. coli

144

culture (2 %) was inoculated into the LB medium and cultured, with shaking (37 0C, 4h). The

145

cell suspension was centrifuged (8000 rpm, room temperature), washed twice with PBS (10 mM,

146

pH 7.4, 1 mL) then re-suspended in PBS (10 mM, pH 7.4) until the optical density at 600 nm

147

remained about 2.0. This suspension was then ready for pathogen capture test

148

different concentrations (4, 6, 8, 10 mg/ml) were dispersed, by ultrasonication (5 min, ultrasonic

149

bath Branson 5800) in PBS (5 mL, 10 mM, pH 7.4). E. coli suspension (5 mL) was then mixed

150

with MNPs suspension (5 mL) by rotary shaking (200 rpm, 370C and aliquot taken periodically

151

to measure the cell concentration after MNPs had been decanted by applying an external 7 ACS Paragon Plus Environment

. The E. coli cells

29, 30

. MNPs, at


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magnetic field. The cell concentration was measured at 600 nm by using an UV/Vis

153

spectrophotometer (V-530, Jasco). An optical density (OD) of 0.1 at 600 nm was equivalent

154

~ 1 .10 × 10 8 cells/ml (CFU).

155 156

The collection efficiency (E, %) at different concentration of MPs@HMCS was calculated according to Eq 1:

E=

157

( A0 − At ) A0

× 100 (Eq. 1)

158

Where A0 is the initial OD, At is the OD after cell capture.

159

The capacity (Q, cells/mg) was calculated according to Eq 2.

Q=

160

161 162 163

( A0 − At ) × 1.1 × 10 8 0 .1 × C

(Eq. 2)

Where A0 is the initial OD, At is the OD after capture and C is the final concentration of particles (mg/mL). S. aureus was also used a model Gram-positive bacterium for the cell capture study. It was

164

grown in Tryptic soy broth (TSB) medium and the study procedure was same as that of E. coli.

165

RESULTS AND DISCUSSION

166

Characterization of HMCS. Chitosan was covalently and hydrophobically modified by a

167

sodium cyanoborohydride reduction of the Schiff base intermediate created between chitosan

168

amino groups and dodecylaldehyde (Scheme 2).


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169 170

Scheme 2. Reaction between chitosan and dodecyl aldehyde.

171 172

Figure 1. FTIR spectra of (a) chitosan and (b) HMCS.

173

The CS and HMCS were characterized by FTIR (Figure 1). The main characteristic peaks of

174

chitosan are at 3375 cm-1 (O–H stretch overlapped with N–H stretch of -NH2 groups), 2882 cm-1

175

(C-H stretch), 1577 cm-1 (N-H bend of primary amino), 1323 cm-1 (C-N stretch), 1154 cm-1

176

(bridge O stretch), and 1094 cm-1 (C-O stretch). The acetyl group absorption is at 1558 cm-1 (N-

177

H of amide II) and 1651 cm-1 (C=O stretch) 29, 31. On the other hand, the IR spectrum of HMCS 9 ACS Paragon Plus Environment


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shows slight differences with a smaller peak at 1577 cm-1 while the methylene (-CH2-) peaks at

179

2924 cm-1, 2855 cm-1, 1420 cm-1 and 1153 cm-1 are larger and sharper. Since the peak intensity is

180

proportional to the extent of substitution

181

hydrophobic tails (C12) was successfully grafted onto the chitosan backbone.

182

31

, the observed intensity increase shows that the

In order to measure the extent of hydrophobic modification in HMCS, elemental analysis was

183

performed.

184

[C6H10O4N(C2H3O)x(H2O)yH1-x]n and [C6H10O4N(C2H3O)x(H2O)y(C12H25)z H1-x-y]n, respectively;

185

where x is the number of acetyl groups or degree of acetylation (DA), z is the number of

186

hydrophobic tails per glucose unit. Based on the element balance, several equations (Eqs. 3 – 5

187

for CS and Eqs. 6 – 8 for HMCS) can be established to solve for x, y and z with the experimental

188

values of the elemental analysis.

189

190

The

empirical

formula

%C =

%N =

for

CS

and

HMCS

are

designated

as

12 × (6 + 2 x) × 100 161 + 42 x + 18 y

(Eq. 3)

14 × 100 161 + 42 x + 18 y

(Eq. 4)

11 + 2 x + 2 y × 100 161 + 42 x + 18 y

(Eq. 5)

%C =

12 × (6 + 2 x + 12 z ) × 100 161 + 42 x + 18 y + 168 z

(Eq. 6)

193

%N =

14 × 100 161 + 42 x + 18 y + 168 z

(Eq. 7)

194

%H =

11 + 2 x + 2 y + 24 z × 100 161 + 42 x + 18 y + 168 z

(Eq. 8)

191

%H =

192

195

Based on the result of elemental analysis (Table S1), the degree of deacetylation (DDA = 100


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196

- DA) of CS and HMCS were calculated to be 72% and 81%, respectively. The increase of DDA

197

of HMCS may be due to some of the N-acetyl groups being removed during the modification

198

reaction. The extent of hydrophobic modification was 11%.

199

Bacterial suspension gelation. In order to investigate their ability to induce gelation of

200

bacterial suspensions, CS and HMCS solution were mixed, separately, with E. coli or S. aureus

201

suspension. The CS/E. coli, CS/S. aureus and HMCS/S. aureus mixtures are free flowing liquids

202

while those for HMCS/E. coli are self-supporting gels that can hold their own weight in the

203

inverted tube as shown (Figure 2). The minimum concentration of HMCS that can induce the

204

gelation of 50% (wet weight/v) of E. coli suspension was determined to be 0.086% (w/v). The

205

very effective gelation of E. coli induced by HMCS is due to the different cell wall structure of

206

Gram positive (S. aureus) and Gram negative (E. coli) bacteria. E. coli has an outer membrane

207

but S. aureus does not. Gelation occurs to E. coli is probably because the hydrophobic tails of

208

HMCS chain anchor within the hydrophobic interior of lipid bilayer of the outer membrane. It

209

has also been demonstrated in previous works that a variety of cell types, including blood cells,

210

endothelial cells and breast cancer cells can be gelled by this approach

211

effect of HMCS on inducing the gelation of E. coli was also demonstrated by the significantly

212

increased steady shear viscosity as shown (Figure 3). Under very low shear rate, the viscosity of

213

HMCS is much higher than CS of the same concentration. This indicates that the intermolecular

214

hydrophobic interactions between hydrophobic tails modified on HMCS contribute to the

215

increase of viscosity. The viscosity of the suspension did not change after mixing CS with E. coli

216

or S. aureus suspension. In contrast, the addition of HMCS to bacterial suspensions significantly

217

increased the viscosity. The 3 to 4 fold higher viscosity increase shows that HMCS has a strong

218

interaction with bacterial cells. However, the viscosity decreased sharply with shear rate which


10, 11

. In addition, the


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indicates the interactions will be disrupted by the high shear rate applied. Based on this observed

220

gelation inducing effect, HMCS was then grafted onto the surface of MNPs, with expectation to

221

capture bacterial cells from a contaminated solution.

222 223 224

Figure 2. Chitosan and HMCS of final concentration 0.086% (w/v) mixed in 50% (wet weight /v) (a) E. coli and (b) S. aureus suspension.

225 226 227 228

Figure 3. Steady-shear rheological data for viscosity with shear stress of CS, HMCS and mixture with bacterial suspensions. The final concentration of bacteria and CS/HMCS are 0.7% and 0.25% (w/v), respectively.

229

Characterization of CS/HMCS grafted MNPs. Commercially available MNPs, used as the

230

magnetic core, were coated with sodium silicate to screen the magnetic dipolar attraction

231

between MNPs and protect the magnetic iron oxide from leaching into the acidic environment. 12 ACS Paragon Plus Environment

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232

Furthermore, a porous and silica-like surface on MNPs was prepared by employing TEOS which

233

would facilitate the surface modification with various functional groups (Scheme 1) 17.

234

The FTIR spectra of the naked MNPs, MNPs@SiO2 and MNPs@SiO2@TEOS are indicated

235

(Figure 4). The peak at 590 cm-1 observed in all of the spectra is related to the Fe – O bonding,

236

and the peak around 3487 cm-1 can be assigned to – OH vibrations on the surface of the particles.

237

The appearance of characteristic bands of Si-O-Si asymmetric/symmetric stretch at 1115 cm-1

238

and 710 cm-1 in curves b and c demonstrates the successful surface modification of MNPs with

239

SiO2 17. In addition, the transmittance intensity ratio of the characteristic peak of silica layer

240

(1115 cm-1) to magnetic nanoparticles core (590 cm-1) significantly increased after coating with

241

TEOS. Furthermore the intensity of the peak around 3487 cm-1, which related to – OH vibrations,

242

is also enhanced after TEOS modification confirming that the MNPs was successfully coated

243

with two shells of silica by sodium silicate and TEOS.

a

% Transmittance

b

4000

244

3500

3000

2500

2000

1500

1000

Fe-O

c

Si-O-Si

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500

-1

Wavenumber (cm )

245 246

Figure 4. FTIR spectra of (a) naked MNPs, (b) MNPs coated with silicate (MNPs@SiO2) and (c) MNPs coated with silicate followed by TEOS (MNPs@SiO2@TEOS).

247

The resultant MNPs@SiO2@TEOS was further surface modified with APTES to obtain

248

amino groups displayed on the particle surface which were finally reacted with GA to obtain the 13 ACS Paragon Plus Environment


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MNPs@GA particles. The number of amino groups and aldehyde groups on the surface of MNPs

250

were determined (Table 1).

251

Table 1 Amino and aldehyde concentration on the surface of MNPs Amount of amino1

Amount of aldehyde2

(1015 molecules/mg)

(1015 molecules/mg)

MNPs@APTES

9.33 ± 0.27

-

MNPs@GA

4.47 ± 0.09

4.68 ± 0.11

Sample

252

1

Ninhydrin assay: calculated with glycine; 2 Sulfuric acid – phenol assay: calculated with GA.

253

The result demonstrates that half of the amino groups on the surface of MNPs@APTES was

254

modified by GA which, in turn, were coupled to CS or HMCS via a Schiff base reaction. The

255

SEM images of naked MNPs, MNPs@GA, MNPs@CS and MNPs@HMCS are shown (Figure

256

S1). The naked MNPs are separate and have a uniform size of about 250 nm. After being surface

257

functionalized with GA, CS or HMCS, the morphology and size did not change significantly.

258

The zeta potential of MNPs, MNPs@CS, MNPs@HMCS were 28.29 ± 2.66, 18.99 ± 3.51 and

259

8.33 ± 1.38 mV, respectively. These particles have the positive net charge at neutral pH and

260

decreased after surface grafted with CS and HMCS. The lower net charge obtained for

261

MNPs@HMCS is probably due to some of the amino groups of CS was modified with alkyl

262

chains.

263

The chitosan and HMCS content in the prepared MNPs@CS and MNPs@HMCS can be

264

determined by using thermo gravimetric analysis (TGA). An obvious weight loss starts at around

265

200oC for all the samples (Figure 5), probably as a result of the removal of bounded water. No

266

appreciable loss occurred for the naked MNPs after 350 oC while the weight loss of MNPs@GA,

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MNPs@CS and MNPs@HMCS is obviously due to the organic compounds that were grafted to

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the naked MNPs. The 2% weight loss difference between CS and HMCS grafted MNPs indicates 14 ACS Paragon Plus Environment

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the amount of HMCS on MNPs is greater than that of CS. Probably, the hydrophobic interactions

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between HMCS molecules would draw more HMCS to be in contact with the activated MNPs so

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that it will have a higher chance to be grafted onto the particles. The magnetic property of

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prepared MNPs was measured by SQUID

273

MNPs and MNPs@HMCS are superparamagnetic with saturation magnetism of 78 and 82 emu/g,

274

respectively (Figure S2). This indicates that the HMCS surface grafting on MNPs did not affect

275

the magnetic properties of Bayoxide MNPs and the high saturation magnetism maintained in

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MNPs@HMCS makes them very responsive to the external magnet employed for collection.

17

. The magnetic hysteresis loop shows that both

100

Weight 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


99

a

98

b c

97 96

d

95 94 100

200

300

400

500

600

700

800

o

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Temperature ( C)

Figure 5. TGA of (a) naked MNPs, (b) MNPs@GA, (c) MNPs@CS and (d) MNPs@HMCS

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Bacterial cells capture. The cell capturing capability of MNPs@HMCS was studied using E.

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coli as a model pathogenic bacterium. Naked MNPs and MNPs@CS have no significant effect

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on decreasing the turbidity of the cell suspension (Figure 6). In contrast, in the presence of

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MNPs@HMCS the decrease of turbidity is dosage dependent. In the presence of 5 mg/mL of

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MNPs@HMCS, the OD600 of E. coli suspension decreased from 1.0 to ~0.25 over 60 min with

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an extremely rapid decrease in OD over the first 20 min due to the removal of cells from solution 15 ACS Paragon Plus Environment


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by MNPs@HMCS. Consequently the efficiency (Q) of MNPs@HMCS to capture E. coli cells

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was calculated from the optical density as measured after 20 min (Figure 7) and was 1.38 x 108

287

cells/mg.

288 289 290 291

Figure 6. Time courses for removing E. coli cells in PBS by MNPs (a) with no MNPs, (b) 5 mg/mL naked MNPs, (c) 5 mg/mL MNPs@CS, (d) 2 mg/mL MNPs@HMCS, (e) 3 mg/mL MNPs@HMCS, (f) 4 mg/mL MNPs@HMCS and (g) 5 mg/mL MNPs@HMCS.

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293 16 ACS Paragon Plus Environment

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Figure 7. Effect of MNPs@HMCS concentration on E. coli collection efficiency at 20 min.

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The removal mechanism of E. coli cells from a contaminated solution by MNPs@HMCS is

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described (Scheme 3). The SEM images of E. coli cells before and after treatment with

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MNPs@HMCS (Figure 8) show that the nanoparticles were attached to the surface of bacterial

298

cell. The hydrophobic tails of grafted HMCS anchor to the membrane of E. coli cells and makes

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the cells magnetically labelled. By applying an external magnetic field, the magnetic particles

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along with E. coli cells can be attracted to the wall of the tube so that a clear solution can be

301

obtained (Figure S3).

302 303 304

Scheme 3. Schematic diagram of E. coli cells removal from contaminated solution by using MNPs@HMCS.

305 306 307

Figure 8. SEM images of (a) E. coli and (b) MNPs@HMCS attached to E. coli cell. (Arrows

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point to the cells). 17 ACS Paragon Plus Environment


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The cell capturing effect of MNPs@HMCS on Gram-positive pathogenic bacteria (S. aureus)

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is shown (Figure 9). Without the addition of any MNPs, the absorbance of S. aureus suspended

311

in PBS decreased with time probably due to the cell autolysis in a solution that contained no

312

nutrients

313

images of MNPs or MNPs@HMCS attached to S. aureus cells was also shown (Figure 10). This

314

indicates that even though there was a strong electrostatic interactions between S. aureus cells

315

and MNPs, the MNPs@CS and MNPs@HMCS were better at capturing S. aureus cells. Since

316

both MNPs@CS and MNPs@HMCS demonstrated a similar cell capturing capability the

317

specific hydrophobic interaction between hydrophobic tails of HMCS and membrane of cells

318

was not seen. This was mainly due to the fact that there is no outer membrane for the Gram-

319

positive bacteria such as S. aureus.

320

MNPs@CS and MNPs@HMCS is as a result of other non-specific interactions between MNPs

321

and cell surface. Even though MNPs@HMCS did not show a significantly better performance

322

than MNPs@CS on removing Gram-positive bacteria, it still demonstrated a high effectiveness

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for removing the Gram-positive bacteria from solution.

26

. All the MNPs decreased the optical density of S. aureus solution and the SEM

Therefore, the excellent capture rate of S. aureus by

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Figure 9. Time course for removing S. aureus cells in PBS by 1 mg/mL MNPs@HMCS.

326 327

Figure 10. SEM images of (a) MNPs + S. aureus and (b) MNPs@HMCS + S. aureus. (Arrows

328

point to the cells).

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CONCLUSIONS

330

Approximately, 11% of the amino groups of chitosan were grafted with dodecyl hydrophobic

331

tail in synthesized HMCS which, not only demonstrated its hemostatic activity but also the

332

gelation of E. coli cells. Both CS and HMCS could be grafted onto the surface of the MNPs via

333

Schiff-base reaction. E. coli cells in the solution could be removed by applying an external

334

magnet field. Naked MNPs and chitosan coated MNPs had no appreciable capturing capacity of

335

E coli cells yet in the presence of MNPs@HMCS, derived from the dodecyl hydrophobic tails,

336

the E. coli capturing capacity was 1.38 x 108 cells/mg. The dodecyl hydrophobic tails on

337

MNPs@HMCS did not show any preferential capturing capacity toward Gram-positive

338

bacterium such as S. aureus cells since both CS and HMCS grafted MNPs had similar capturing

339

capacity. The HMCS grafted MNPs therefore can be used for the collection of liposome or other

340

Gram-negative bacteria which have their membrane structure directly exposed to the solution. In

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addition, the prepared MNPs@HMCS may be used to remove the biofilm formed by Gram-

342

negative bacteria at a specific site by applying an external magnetic field to manipulate the 19 ACS Paragon Plus Environment


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MNPs for removal of the biomass.

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ASSOCIATED CONTENT

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Supporting Information Available

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The data of elemental analysis of CS and HMCS, SEM images of MNPs@HMCS, the magnetic

347

hysteresis loop of naked MNPs, MNPs@HMCS at 298 K and figure of E. coli treatment. This

348

information is available free of charge via the Internet at http://pubs.acs.org/.

349

AUTHOR INFORMATION

350

Corresponding Author

351

*C.K, Lee. E-mail: [email protected]. Tel.: +886-2-27376629. Fax: +886-2-27376644.

352

References

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Hydrophobically Modified Chitosan-Grafted Magnetic Nanoparticles

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