Subscriber access provided by CMU Libraries - http://library.cmich.edu
Article
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
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Industrial & Engineering Chemistry Research is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 22
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
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
Industrial & Engineering Chemistry Research
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 2 of 22
23
deacetylation. Due to its biocompatibility, antimicrobial activity, wound healing property, and
24
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
35
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
41
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
Page 3 of 22
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
46 47
Industrial & Engineering Chemistry Research
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.
52
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
55
metal ion adsorption, enzyme immobilization, affinity proteins adsorption and many other
56
applications
57
able to specifically capture the vancomycin-resistant enterococci and Gram-positive bacteria at
58
an ultralow concentration
59
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
62
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
66
Staphylococcus aureus were decontaminated using the synthesized MNPs@HMCS.
67
EXPERIMENTAL SECTION
68
Materials. Chitosan, from shrimp shells (MM ~ 512 kDa, calculated by intrinsic viscosity 3 ACS Paragon Plus Environment
Industrial & Engineering Chemistry Research
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
69
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
71
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
76
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
79
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
Page 4 of 22
Page 5 of 22
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
Industrial & Engineering Chemistry Research
17
.
92
surface modification of MNPs for CS or HMCS was based on that reported in the literature
93
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.
104
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
5 ACS Paragon Plus Environment
Industrial & Engineering Chemistry Research
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
114 115 116
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
6 ACS Paragon Plus Environment
Page 6 of 22
Page 7 of 22
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
Industrial & Engineering Chemistry Research
129
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
Industrial & Engineering Chemistry Research
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 8 of 22
152
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).
8 ACS Paragon Plus Environment
Page 9 of 22
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
Industrial & Engineering Chemistry Research
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
Industrial & Engineering Chemistry Research
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 10 of 22
178
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
10 ACS Paragon Plus Environment
Page 11 of 22
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
Industrial & Engineering Chemistry Research
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
11 ACS Paragon Plus Environment
10, 11
. In addition, the
Industrial & Engineering Chemistry Research
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 12 of 22
219
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
Page 13 of 22
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
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
Industrial & Engineering Chemistry Research
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
Industrial & Engineering Chemistry Research
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 14 of 22
249
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,
267
MNPs@CS and MNPs@HMCS is obviously due to the organic compounds that were grafted to
268
the naked MNPs. The 2% weight loss difference between CS and HMCS grafted MNPs indicates 14 ACS Paragon Plus Environment
Page 15 of 22
269
the amount of HMCS on MNPs is greater than that of CS. Probably, the hydrophobic interactions
270
between HMCS molecules would draw more HMCS to be in contact with the activated MNPs so
271
that it will have a higher chance to be grafted onto the particles. The magnetic property of
272
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
276
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
Industrial & Engineering Chemistry Research
99
a
98
b c
97 96
d
95 94 100
200
300
400
500
600
700
800
o
277 278
Temperature ( C)
Figure 5. TGA of (a) naked MNPs, (b) MNPs@GA, (c) MNPs@CS and (d) MNPs@HMCS
279
Bacterial cells capture. The cell capturing capability of MNPs@HMCS was studied using E.
280
coli as a model pathogenic bacterium. Naked MNPs and MNPs@CS have no significant effect
281
on decreasing the turbidity of the cell suspension (Figure 6). In contrast, in the presence of
282
MNPs@HMCS the decrease of turbidity is dosage dependent. In the presence of 5 mg/mL of
283
MNPs@HMCS, the OD600 of E. coli suspension decreased from 1.0 to ~0.25 over 60 min with
284
an extremely rapid decrease in OD over the first 20 min due to the removal of cells from solution 15 ACS Paragon Plus Environment
Industrial & Engineering Chemistry Research
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 16 of 22
285
by MNPs@HMCS. Consequently the efficiency (Q) of MNPs@HMCS to capture E. coli cells
286
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.
292
293 16 ACS Paragon Plus Environment
Page 17 of 22
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
294
Industrial & Engineering Chemistry Research
Figure 7. Effect of MNPs@HMCS concentration on E. coli collection efficiency at 20 min.
295
The removal mechanism of E. coli cells from a contaminated solution by MNPs@HMCS is
296
described (Scheme 3). The SEM images of E. coli cells before and after treatment with
297
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
299
the cells magnetically labelled. By applying an external magnetic field, the magnetic particles
300
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
308
point to the cells). 17 ACS Paragon Plus Environment
Industrial & Engineering Chemistry Research
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 18 of 22
309
The cell capturing effect of MNPs@HMCS on Gram-positive pathogenic bacteria (S. aureus)
310
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
323
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
324 18 ACS Paragon Plus Environment
Page 19 of 22
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
325
Industrial & Engineering Chemistry Research
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).
329
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
341
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
Industrial & Engineering Chemistry Research
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 22
343
MNPs for removal of the biomass.
344
ASSOCIATED CONTENT
345
Supporting Information Available
346
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
353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374
1. Liu, C.-G.; Desai, K. G. H.; Chen, X.-G.; Park, H.-J., Linolenic Acid-Modified Chitosan for Formation of Self-Assembled Nanoparticles. J. Agric. Food Chem. 2004, 53, 437. 2. Mourya, V. K.; Inamdar, N. N.; Tiwari, A., Carboxymethyl chitosan and its applications. Adv. Mater. Lett. 2010, 1, 11. 3. Zhu, A.; Chan-Park, M. B.; Dai, S.; Li, L., The aggregation behavior of Ocarboxymethylchitosan in dilute aqueous solution. Colloids Surf., B 2005, 43, 143. 4. Zhang, J.; Chen, X. G.; Li, Y. Y.; Liu, C. S., Self-assembled nanoparticles based on hydrophobically modified chitosan as carriers for doxorubicin. Nanomedicine 2007, 3, 258. 5. Na, K.; Bum Lee, T.; Park, K.-H.; Shin, E.-K.; Lee, Y.-B.; Choi, H.-K., Self-assembled nanoparticles of hydrophobically-modified polysaccharide bearing vitamin H as a targeted anticancer drug delivery system. Eur. J. Pharm. Sci. 2003, 18, 165. 6. Liu, C.-G.; Desai, K. G. H.; Chen, X.-G.; Park, H.-J., Preparation and Characterization of Nanoparticles Containing Trypsin Based on Hydrophobically Modified Chitosan. J. Agric. Food Chem. 2005, 53, 1728. 7. Almada M Fau - Burboa, M. G.; Burboa Mg Fau - Robles, E.; Robles E Fau - Gutierrez, L. E.; Gutierrez Le Fau - Valdes, M. A.; Valdes Ma Fau - Juarez, J.; Juarez, J., Interaction and cytotoxic effects of hydrophobized chitosan nanoparticles on MDA-MB-231, HeLa and Arpe-19 cell lines. Curr. Med. Chem. 2014, 14, 692. 8. Chiu, Y. L.; Ho, Y. C.; Chen, Y. M.; Peng, S. F.; Ke, C. J.; Chen, K. J.; Mi, F. L.; Sung, H. W., The characteristics, cellular uptake and intracellular trafficking of nanoparticles made of hydrophobically-modified chitosan. J Control Release 2010, 146, 152. 9. St. Dennis, J. E.; Meng, Q.; Zheng, R.; Pesika, N. S.; McPherson, G. L.; He, J.; Ashbaugh, H. 20 ACS Paragon Plus Environment
Page 21 of 22
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
375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418
Industrial & Engineering Chemistry Research
S.; John, V. T.; Dowling, M. B.; Raghavan, S. R., Carbon microspheres as network nodes in a novel biocompatible gel. Soft Matter 2011, 7, 4170. 10. Dowling, M. B.; Kumar, R.; Keibler, M. A.; Hess, J. R.; Bochicchio, G. V.; Raghavan, S. R., A self-assembling hydrophobically modified chitosan capable of reversible hemostatic action. Biomaterials 2011, 32, 3351. 11. Javvaji, V.; Dowling, M. B.; Oh, H.; White, I. M.; Raghavan, S. R., Reversible gelation of cells using self-assembling hydrophobically-modified biopolymers: towards self-assembly of tissue. Biomaterials Science 2014, 2, (7), 1016. 12. Ashtari, P.; He, X.; Wang, K.; Gong, P., An efficient method for recovery of target ssDNA based on amino-modified silica-coated magnetic nanoparticles. Talanta 2005, 67, 548. 13. Berensmeier, S., Magnetic particles for the separation and purification of nucleic acids. Appl. Microbiol. Biotechnol. 2006, 73, 495. 14. Lai, Y.; Yin, W.; Liu, J.; Xi, R.; Zhan, J., One-Pot Green Synthesis and Bioapplication of lArginine-Capped Superparamagnetic Fe3O4 Nanoparticles. Nano Lett. 2010, 5, 302. 15. Markova, Z.; Siskova, K.; Filip, J.; Safarova, K.; Prucek, R.; Panacek, A.; Kolar, M.; Zboril, R., Chitosan-based synthesis of magnetically-driven nanocomposites with biogenic magnetite core, controlled silver size, and high antimicrobial activity. Green Chem. 2012, 14, 2550. 16. Grüttner, C.; Rudershausen, S.; Teller, J., Improved properties of magnetic particles by combination of different polymer materials as particle matrix. J. Magn. Magn. Mater. 2001, 225, 1. 17. Lin, Z.-A.; Zheng, J.-N.; Lin, F.; Zhang, L.; Cai, Z.; Chen, G.-N., Synthesis of magnetic nanoparticles with immobilized aminophenylboronic acid for selective capture of glycoproteins. J. Mater. Chem. 2011, 21, 518. 18. Zhang, X.; Niu, H.; Pan, Y.; Shi, Y.; Cai, Y., Chitosan-Coated Octadecyl-Functionalized Magnetite Nanoparticles: Preparation and Application in Extraction of Trace Pollutants from Environmental Water Samples. Anal. Chem. 2010, 82, 2363. 19. Yang, L.; Guo, C.; Jia, L.; Xie, K.; Shou, Q.; Liu, H., Fabrication of biocompatible temperature- and pH-responsive magnetic nanoparticles and their reversible agglomeration in aqueous milieu. Ind. Eng. Chem. Res. 2010, 49, 8518. 20. Wu, Y.; Wang, Y.; Luo, G.; Dai, Y., In situ preparation of magnetic Fe3O4-chitosan nanoparticles for lipase immobilization by cross-linking and oxidation in aqueous solution. Bioresour. Technol. 2009, 100, 3459. 21. Gu, H.; Ho, P.-L.; Tsang, K. W. T.; Wang, L.; Xu, B., Using Biofunctional Magnetic Nanoparticles to Capture Vancomycin-Resistant Enterococci and Other Gram-Positive Bacteria at Ultralow Concentration. J. Am. Chem. Soc. 2003, 125, 15702. 22. El-Boubbou, K.; Gruden, C.; Huang, X., Magnetic glyco-nanoparticles: A unique tool for rapid pathogen detection, decontamination, and strain differentiation. J. Am. Chem. Soc. 2007, 129, 13392. 23. Geng, X.; Kwon, O.-H.; Jang, J., Electrospinning of chitosan dissolved in concentrated acetic acid solution. Biomaterials 2005, 26, 5427. 24. Desbrières, J.; Martinez, C.; Rinaudo, M., Hydrophobic derivatives of chitosan: Characterization and rheological behaviour. Int. J Biol. Macromolecules 1996, 19, 21. 25. Drochioiu, G., I. ; Mangalagiu, E.; Avram, K.; Popa, A. C.; Druta, D. a. I., Cyanide Reaction with Ninhydrin: Elucidation of Reaction and Interference Mechanisms. Anal. Sci. 2004, 20, 1443. 21 ACS Paragon Plus Environment
Industrial & Engineering Chemistry Research
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
419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435
26. Chen, T.; Wang, R.; Xu, L. Q.; Neoh, K. G.; Kang, E.-T., Carboxymethyl ChitosanFunctionalized Magnetic Nanoparticles for Disruption of Biofilms of Staphylococcus aureus and Escherichia coli. Ind. Eng. Chem. Res. 2012, 51, 13164. 27. Kong, M.; Chen, X. G.; Liu, C. S.; Liu, C. G.; Meng, X. H.; Yu le, J., Antibacterial mechanism of chitosan microspheres in a solid dispersing system against E. coli. Colloids Surf., B 2008, 65, 197. 28. Szegedi, A.; Popova, M.; Goshev, I.; Klebert, S.; Mihaly, J., Controlled drug release on amine functionalized spherical MCM-41. J. Solid State Chem. 2012, 194, 257. 29. Liu, X. F.; Guan, Y. L.; Yang, D. Z.; Li, Z.; Yao, K. D., Antibacterial action of chitosan and carboxymethylated chitosan. J. Appl. Polym. Sci. 2001, 79, 1324. 30. Wang, R.; Neoh, K. G.; Shi, Z.; Kang, E.-T.; Tambyah, P. A.; Chiong, E., Inhibition of escherichia coli and proteus mirabilis adhesion and biofilm formation on medical grade silicone surface. Biotechnol. Bioeng. 2012, 109, 336. 31. Li, Y.-Y.; Chen, X.-G.; Yu, L.-M.; Wang, S.-X.; Sun, G.-Z.; Zhou, H.-Y., Aggregation of hydrophobically modified chitosan in solution and at the air-water interface. J. Appl. Polym. Sci. 2006, 102, 1968.
436 437 438 439 440 441
Page 22 of 22
For Table of Contents Only
442
22 ACS Paragon Plus Environment