Accepted Manuscript Title: Optimized synthesis of O-carboxymethyl-N,N,N-trimethyl chitosan Author: V. Patrulea Lee Ann Applegate V. Ostafe O. Jordan G. Borchard PII: DOI: Reference:
S0144-8617(14)01197-7 http://dx.doi.org/doi:10.1016/j.carbpol.2014.12.014 CARP 9504
To appear in: Received date: Revised date: Accepted date:
20-10-2014 12-12-2014 16-12-2014
Please cite this article as: Patrulea, V., Applegate, L. A., Ostafe, V., Jordan, O., and Borchard, G.,Optimized synthesis of O-carboxymethyl-N,N,N-trimethyl chitosan, Carbohydrate Polymers (2014), http://dx.doi.org/10.1016/j.carbpol.2014.12.014 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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solvent precipitation
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Improved chitosan O-carboxymethylation up to a degree of 80%, based on Omethyl-free N-trimethyl chitosan
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Improved chitosan N-trimethylation through optimization of reaction time and
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Cytocompatibility of
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Highlights:
the CMTMC chitosan derivative towards human
fibroblasts
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Optimized synthesis of O-carboxymethyl-N,N,N-trimethyl chitosan
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V. Patruleaa,b,c, Lee Ann Applegated, V. Ostafeb,c , O. Jordana, G. Borcharda,*
11
a
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Quai Ernest Ansermet 30, 1211, Geneva, Switzerland
13
b
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Timisoara, 300115, Romania
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c
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Oituz 4, Timisoara 300086, Romania
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d
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Medicine, EPCR/02/ch. Croisettes 22, 1066 Epalinges, Switzerland
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*
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E-mail address:
[email protected] 21
Abstract
22
We present here the synthesis of a highly O-carboxymethylated chitosan derivative.
23
First, an improved protocol for the two-step synthesis of N-trimethyl chitosan (TMC)
24
from chitosan was developed, yielding a maximum degree of quaternisation (DQ) of
25
up to 46.6%. Successively, the chitosan derivative O-carboxymethyl-N-trimethyl
26
chitosan (CMTMC) was synthesized from the TMC obtained by applying an optimized
27
synthesis pathway. In contrast to previous reports, the optimized protocol was shown
28
to yield very high rates (>85%) of O-carboxymethylation of CMTMC, as shown by 1H-
29
NMR and heteronuclear single quantum correlation (1H-13C HSQC). Finally, in vitro
30
cytocompatibility (viability >80%) of the polymer was demonstrated using human
31
fibroblasts.
32
Highlights:
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West University of Timisoara, Department of Biology-Chemistry, Pestalozzi 16,
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West University of Timisoara, Advanced Environmental Research Laboratories,
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Corresponding author. Tel.: +41 22 379 6945
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University Hospital of Lausanne (CHUV-UNIL), Department of Musculoskeletal
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Improved chitosan N-trimethylation through optimization of reaction time and solvent precipitation
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School of Pharmaceutical Sciences, University of Geneva, University of Lausanne,
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Improved chitosan O-carboxymethylation up to a degree of 80%, based on Omethyl-free N-trimethyl chitosan
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Cytocompatibility of
the CMTMC chitosan derivative towards human
fibroblasts chitosan,
chitosan
derivatives,
drug/gene
delivery,
methylation,
Keywords:
40
quaternization.
41
Chemical compounds: chitosan (PubChem CID: 21896651); chloroacetic acid
42
(PubChem CID: 300); methyl iodide (PubChem CID: 6328); formic acid (PubChem
43
CID: 284); formaldehyde (PubChem CID: 712); isopropanol (PubChem CID: 3776).
44
Abbreviations: TMC: N,N,N-trimethyl chitosan; CMTMC: O-carboxymethyl-N,N,N-
45
trimethyl chitosan; DQ: degree of quaternization.
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1. Introduction
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Chitosan, a biopolymer used in numerous medical applications, is obtained
48
through partial deacetylation of chitin (Sieval et al., 1998). Chitosan has been widely
49
used in medical applications, wastewater treatment, textile industry, cosmetics and
50
agriculture, because of its unique properties. Indeed, chitosan is a biodegradable,
51
biocompatible,
52
antimicrobial activities (Jayakumar et al., 2010; Lee et al., 2014; Muzzarelli, 2009;
53
Rinaudo, 2008), and is also an efficient biosorbent useful for environmental purposes
54
(Patrulea et al., 2013). However, the application of chitosan is limited due to its poor
55
solubility at physiological pH, which in turn decreases efficiency of drug delivery
56
below pH 6 (Sieval et al., 1998). In this view, several methods were investigated to
57
improve chitosan solubility. The preferred approach to increase chitosan solubility at
58
neutral pH is to introduce permanent charges into the chitosan backbone. In order to
59
obtain a water-soluble chitosan derivative, Domard et al. (1986) prepared
60
quaternized chitosan through methylation (Domard et al., 1986), while Chen and Park
61
(2003) developed carboxymethylated chitosan, and Holme and Perkin (1997)
62
proposed sulfonated chitosan.
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non-toxic
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and
polymer,
with
antibacterial
and
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N,N,N-Trimethyl chitosan chloride (TMC) is a positively charged chitosan
64
derivative soluble at neutral pH. It has been shown that TMC has an enhanced
65
solubility, promotes intestinal absorption of peptides and drugs, and has
66
mucoadhesive properties (Amidi et al., 2006; Polnok et al., 2004). Nanoparticles
67
based on TMC are used as delivery systems for drugs, DNA or RNA therapeutics. 3 Page 3 of 18
Domard et al. and later Sieval et al. prepared TMC by treating chitosan with N-
69
methyl-2-pyrrolidone followed by reaction with methyl iodide in the presence of
70
sodium hydroxide and sodium iodide (Domard et al., 1986; Sieval et al., 1998). The
71
reaction was carried out in several steps in order to obtain a higher degree of
72
substitution (up to 64%), but drawbacks were reported, such as a reduced molecular
73
weight attributed to chain scission, decreased solubility, and uncontrolled degree of
74
trimethylation leading to O-methylation (Verheul et al., 2008). Generally, during
75
quaternization, O-methylation may occur, leading to merely loosely controlled
76
polymer characteristics. In order to avoid O-methylation, Muzzarelli et al. reported on
77
a method for N-dimethyl-chitosan (DMC) formation followed by synthesis of TMC with
78
formaldehyde and borohydride (Muzzarelli & Tanfani, 1985). The same principle was
79
used by Verheul et al. (2008) and Xu et al. (2010) by switching from borohydride to
80
using formic acid.
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N,O-Carboxymethyl-chitosan (CMC) has drawn much attention due to its
82
improved solubility in water. CMC is a polyelectrolyte derivative, which contains
83
carboxymethyl substituents at carboxyl and amino sites. CMC is a water-soluble
84
polymer, with its solubility being related to the degree of substitution (George &
85
Abraham, 2006; Muzzarelli, 1988). Some authors reported that CMC is biocompatible
86
and mucoadhesive polymer, which does not induce immune responses (Jayakumar
87
et al., 2010; Yin et al., 2007) and is of low toxicity (Tokura et al., 1996). Such
88
characteristics turn CMC into a unique platform for grafting peptides, proteins or
89
drugs for effective delivery (Wang et al., 2010; Wang et al., 2013), cell delivery
90
vehicle for in situ bone tissue engineering (Mishra et al., 2011) and safe parenterally
91
applied system (Fu et al., 2011). In view of functionalizing chitosan with high amount
92
of peptide for increased bioactivity, chitosan bearing a high number of O-
93
carboxymethyl alkyl groups is a highly desirable product.
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The aim of this study was therefore to develop a water-soluble O-
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carboxymethyl-N,N,N-trimethyl chitosan (CMTMC) with a degree of substitution
96
higher than previously reported, based on TMC without O-methylation, as shown in
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Fig. 1. To this end, optimization of each synthesis step – N-trimethylation and O-
98
carboxymethylation - was carried out.
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Fig. 1. Optimized synthesis of O-carboxymethyl-N,N,N-trimethyl chitosan (CMTMC).
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2. Materials and methods
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2.1 Materials
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Chitosan (79% degree of deacetylation, ChitoClear Cg10, 7–15 mPa*s) was
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bought from Primex (Siglufjordur, Iceland). Chloroacetic acid, methyl iodide (CH3I)
105
(99%), N-methyl-2-pyrrolidone (NMP) (99%), deuterium oxide (D2O), deuterium
106
chloride (DCl), deuterium oxide supplemented with 0.05% 3-(trimethylsilyl)propionic-
107
2,2,3,3-d4 acid and sodium salt (TSP) were purchased from Sigma–Aldrich (Buchs,
108
Switzerland). Sodium hydroxide (98.5%) and diethylether (99.5%) were obtained
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from Acros Organics (Geel, Belgium). Formic acid (85%) was bought from Reactolab
110
(Servion, Switzerland) and formaldehyde (37%) was ordered from Merck (Darmstadt,
111
Germany). Ethanol (99.9%) and isopropanol (99.9%) were obtained from Fischer
112
(Wallingford, UK). Dulbecco`s modified Eagle medium (DMEM) and all other
113
reagents were purchased from Sigma–Aldrich (Buchs, Switzerland).
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2.2 Methods
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O-Carboxymethyl-N,N,N-trimethyl chitosan (CMTMC) synthesis CMTMC was obtained by O-carboxymethylation of N-trimethyl chitosan (TMC).
117
TMC was synthesized in two ways, either by a “conventional” 1-step method ("TMC
118
1"), or an “optimized” 2-step method ("TMC 2"). CMTMC was further obtained by
119
carboxymethylation of TMC 2 based either on a “conventional” protocol previously
120
used in our group (Hansson et al., 2012a) yielding a product designated "CMTMC 1",
121
or using a new method to increase the substitution degree and potentially enhance
122
drug, peptide or protein grafting ("CMTMC 2").
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Fig. 2. Scheme of the experiments performed for (a) TMC 1 synthesis by
125
conventional 1-step method, (b) TMC 2 synthesis by optimized 2-step method and (c)
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O-carboxymethylation of TMC 2.
127
2.2.1. Conventional synthesis method – TMC 1
128
For the conventional synthesis, TMC was synthesized through nucleophilic
129
substitution with CH3I as a reagent, sodium iodide as catalyst and sodium hydroxide
130
(NaOH) as base with NMP as solvent, modifying the protocol described by Hansson
131
et al. (Hansson et al., 2012b). We evaluated the effects of reaction time and solvent
132
used for precipitation on quaternization.
133
Briefly, 4.0 g of chitosan and 9.6 g of NaI (molar ratio 4:1) were added to 160
134
mL pre-warmed NMP at 60 °C. After 30 min of reaction, the pH of the solution was
135
adjusted to a value of 10 using 15% (w/v) NaOH and left to react for 15 min. Later, 24 6 Page 6 of 18
mL of CH3I (chitosan:CH3I molar ratio of 1:2) were added and left to react for the
137
desired time (varying, see below). The solution was filtered through a P3 glass filter
138
under vacuum in order to remove any insoluble residues. Then, the solution was
139
precipitated by adding 5 volumes of ethanol and diethylether at different ratios
140
(varying, see below). Subsequently, TMC was separated from the supernatant by
141
centrifugation (Beckmann Avanti TM 30, UK) at 3220 x g for 15 min. The precipitate
142
was dissolved in 10% NaCl and then stirred overnight at room temperature.
143
Furthermore, dialysis (Spectra/Por 4, cut-off 12-14 kDa, Spectra, USA) was
144
performed for 3 days with water changes twice daily. Samples were lyophilized
145
(Christ Alpha 2-4 LD Plus, Switzerland), which was followed by analysis.
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In order to study the effect of reaction time, we left reactions for 20, 40, 60,
147
100, 140 min or overnight. After filtration, TMC was precipitated in a 1:1 mixture of
148
ethanol and diethylether. We also investigated the effect of ethanol:diethylether ratio
149
(1:0; 1:1; 2:1; 3:1; 4:1; 5:1; 1:2; 1:3; 1:4 or 1:5) used for precipitation, for a given
150
reaction time of 140 min.
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2.2.2. Optimized method – TMC 2
The optimized synthesis of TMC was carried out in two steps: first by the
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Eschweiler-Clarke reaction, treating chitosan with formaldehyde in the presence of
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formic acid using a protocol modified from Verheul et al. (2008) and second by
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treating the obtained N-dimethyl-chitosan (DMC) with CH3I in order to obtain TMC 2,
156
using the best reaction time and precipitation solvent ratio determined for TMC 1.
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Briefly, 2 g of chitosan were added to 6 mL of formic acid, 8 mL of
158
formaldehyde and 36 mL of distilled water. This reaction was carried out at 70 °C
159
under reflux (Liebig condenser) for 118 h. Afterwards, the volume was reduced with a
160
Rotavapor and the pH was subsequently adjusted to 12 using 1M NaOH. The
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precipitate was isolated by paper filtration, followed by washing with water in order to
162
remove any impurities and un-reacted formaldehyde. Filtrated N-dimethyl-chitosan
163
(DMC) was suspended in water at a concentration of 2% (w/v) and the pH adjusted to
164
4 using 1 M HCl and further stirred until complete DMC dissolution. The solution was
165
filtered and dialyzed for 3 days, with water changes twice daily. Then the product was
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lyophilized for 2 days and analyzed.
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1 g of dried DMC was solubilized in 160 mL of water under stirring at room
168
temperature. The pH was adjusted to 11 by drop-wise addition of 1M NaOH until gel
169
formation occurred. This step was followed by washing twice with water and three
170
times with acetone through a filter. Then, dried precipitate was suspended in 50 mL
171
NMP and the dispersion was stirred at 70 °C under reflux conditions until the
172
complete dissolution of the chitosan derivative (DMC). Next, CH3I was added at a
173
ratio of 1:25 to the reaction medium and left for 140 min to complete the reaction.
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Subsequently, precipitation with ethanol:diethylether (4:1) and centrifugation at 3220
175
x g for 15 min were performed. Dialysis was done for 3 days. In order to obtain a
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better ion exchange, the first day of dialysis was done against a 1% NaCl solution in
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water and the last 2 days against water, changing dialysis media twice per day. The
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obtained TMC was lyophilized and analyzed as described below.
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2.2.3. CMTMC synthesis: TMC carboxymethylation
CMTMC obtained with conventional carboxymethylation protocol – CMTMC 1
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A quantity of 0.2 g of dried TMC 2 was suspended in 20 mL NMP and left to
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stir overnight at ambient temperature. The pH was adjusted to 10 with 15% NaOH,
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and kept constant by NaOH addition for 1 h. Then, 20 equivalents of mono-
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chloroacetate were added to the reaction and the pH was kept constant during 3 h of
185
the reaction. After 3 h, reaction product was precipitated in a mixture of
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ethanol:diethylether (4:1). After centrifugation at 3220 x g for 15 min, 10 mL of Milli-Q
187
water were added to dissolve the precipitate and the pH was adjusted to 5 with 1M
188
HCl. The solution was dialyzed for 3 days, changing water twice per day, and
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lyophilized samples were analyzed.
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CMTMC obtained with new carboxymethylation protocol – CMTMC 2
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An amount of 0.1 g of dried TMC was suspended in 10 mL of NMP or
192
isopropanol as solvent and left to stir overnight at 45 °C. During 20 min, under
193
continuous stirring, 0.5 mL of 50% NaOH was slowly added to the reaction mixture
194
that was stirred for another 45 min. Subsequently, 0.12 g of mono-chloroacetate
195
(molar ratio 1:3 to TMC) was added during 25 min and reaction was left for 3 h at 60
196
°C under a nitrogen atmosphere in order to avoid TMC degradation. Afterwards, pH
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was adjusted to neutral with 1M HCl followed by filtration, washing with 70% ethanol
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and subsequently with anhydrous ethanol. CMTMC precipitate was dried under 8 Page 8 of 18
vacuum, dissolved in distilled water and dialyzed for 3 days against water, changing
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dialysis medium twice per day. The dialyzed CMTMC 2 was lyophilized and later
201
analyzed.
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2.3. Characterization of polymers
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2.3.1. Determination of the degree of substitution
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1
H-NMR and
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C-NMR spectra were recorded with Varian Gemini 300 MHz or
500 MHz (Agilent Technologies, Santa Clara, CA, USA). Chemical shifts are reported
206
as part per million (ppm) at room temperature. The samples were dissolved in 1%
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DCl/D2O with 0.05% trimethylsilyl propionic acid (TSP) as internal standard for
208
=0.00 ppm. The protons in the chitosan derivatives were attributed according to
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literature (Hansson et al., 2012b; Heuking et al., 2009; Polnok et al., 2004) as follows:
210
=2.0 ppm (COCH3); =2.8 (NHCH3); =3.0 (N(CH3)2); =3.3 (N+(CH3)3); =4.15-4.5
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(OCH2COOH) and =5-5.7 (H-1).
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The degrees of monomethylation (MM), dimethylation (Luca et al., 2010),
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trimethylation (Howling et al., 2001) and carboxymethylation (CM) were calculated
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with the methods previously described, using Eq. (1-4) as shown below (Polnok et
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al., 2004):
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(1) (2) (3) (4)
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[CH3] is the value of the integral for the MM functions at 2.8 ppm; [(CH3)2] is
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the integral of dimethyl amino DM at 3.0 ppm, [(CH3)3] is the integral for trimethyl
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amino functions at 3.3 ppm, [(OCH2—COOH)] represents the integral for
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carboxymethyl groups at 4.15-4.5 ppm and [H] is the value for the integral attributed
224
to H-1 peaks between 5 and 5.7 ppm.
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2.3.2. Cytotoxicity Test WST-1
In vitro toxicity of CMTMC was evaluated using the cell proliferation reagent
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WST-1 (Roche, Switzerland) on human dermal fibroblasts (provided by Centre
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Hospitalier Universitaire Vaudois under Ethics Committee Protocol #62/07,
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Lausanne, Switzerland). Human dermal fibroblasts were seeded in a 96-well plate at
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an initial seeding density of 5×103 cells per well and incubated for 48 h at 37 °C and
231
5% CO2. Afterwards, the culture medium was replaced by a polymer solution in
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DMEM with concentrations of either: 1 mg mL-1, 0.5 mg mL-1 or 0.1 mg mL-1. Cells
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were incubated for 0, 2, 4, 7 and 10 days. Positive (DMEM) and negative controls
234
(sodium dodecyl sulfate (SDS) 1%) were used. Polymer suspension was replaced
235
every 3 days. Subsequently, polymer suspensions were aspirated and replaced by
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100 µL of WST-1 (diluted 1:10 in DMEM) and incubated for 0.5 h to 4 h. Absorbance
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(BioTek Microplate Reader, GmbH, Luzern, Switzerland) was measured at
238
wavelengths 450 and 690 nm according to Fisichella et al. (2009).
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3.
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3.1. TMC characterization
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Results and discussions
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TMC 1 synthesis through direct trimethylation of chitosan with iodomethane
242
led to O-methylation, as shown by NMR peak at =3.4 ppm (Fig. 3A), which can be
243
attributed to O-CH3. This is in agreement with uncontrolled O-methylation at C-3 and
244
C-6 as previously reported (Verheul et al., 2008). A similar process was followed by
245
Li et al. (2010), which is likely to result in non-selective O- and N-trimethylation.
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Hence,
247
carboxymethylation would be difficult to control.
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Table 1.
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Increase of the degree of trimethylation during the reaction (n=3).
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the
degree
Reaction time (min)
of
quaternization
(DQ)
and
in
turn
the
degree
of
Degree of quaternization (%)
100
24.5±1.2
140
36.8±9.3
overnight
22.7±3.3
250
10 Page 10 of 18
Varying reaction time (from 40, 60, 100, 140 and overnight) influences DQ
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(Table 1). For 40 and 60 min (data not shown), the DQ achieved was about 20%.
253
While increasing reaction time to 100 and 140 min, DQ increased linearly reaching a
254
plateau after 140 min. These results are in good agreement with Kean et al., who
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showed that the DQ of TMC increased linearly until 120 min of reaction reaching a
256
plateau (Kean et al., 2005). In addition, we observed that changing the solvent ratio
257
influenced the DQ. The best DQ (41±9.8%) was obtained with a 4:1 solvent ratio.
258
Higher and lower ratios yielded DQ in the range of 17 to 24%, by changing solvent
259
ratio ethanol:diethylether (1:0; 1:1; 2:1; 3:1; 5:1; 1:2; 1:3; 1:4; 1:5).
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For TMC 2 optimized synthesis, we used the reaction time (140 min) and the
261
solvent ratio (4:1) that lead to highest degrees of TMC 1 quaternization. We obtained
262
a higher degree of substitution (46.6%) in the absence of O-methylation at C-3 and
263
C-6, as confirmed by NMR (Fig. 3). Moreover, chain scission was prevented (size
264
exclusion chromatography data, not shown). This method is expected to control the
265
DQ without altering the structural properties of the glucosamine units (Verheul et al.,
266
2008).
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Fig. 3. 1H-NMR spectra for TMC 1 (A) and for TMC 2 (B) in D2O.
269
In Fig. 3 are presented chemical shifts for TMC 1 and TMC 2. The signal
270
intensity present in TMC 1 at 3.4 ppm (peak 8) demonstrates that O-methylation 11 Page 11 of 18
occurred during the reaction. In contrast, spectra (B) of TMC 2 obtained through the
272
optimized method shows O-methylation-free TMC. As a result, DQ increased from
273
29.4% for TMC 1 to 46.6% for TMC 2. This increased quaternization was also related
274
to an improvement of solubility and is considered to be advantageous for
275
complexation with anionic polymers in order to form nanoparticles, or to carry drug or
276
nucleic acids for pharmaceutical applications.
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3.1.1. CMTMC characterization
Concerning carboxymethylation, Hansson et al. used chloroacetic acid in NMP
279
under basic conditions (pH 10). By varying reaction time, they substituted up to 27%
280
carboxymethyl groups (Hansson et al., 2012b). This relatively low value may be due
281
to previous O-methylation at C-3 and C-6, resulting in a low availability of OH groups
282
for carboxymethylation (Fig. 4A). Using NMP, we also observed a very low degree of
283
substitution (data not shown). Switching reaction medium to isopropanol led to better
284
results, achieving degrees of O-carboxymethylation in extent of 85% (Fig. 4B) at an
285
overall yield of 74%. This might be related to a better conformation of TMC in
286
isopropanol offering higher accessibility to the reaction sites.
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287 288
Fig. 4. 1H-NMR spectra for CMTMC 1 (A) and CMTMC 2 (B). Red bullets correspond
289
to O-methylated C-3 and C-6.
12 Page 12 of 18
The high degree of O-carboxymethylation was obtained by using a strong
291
basic medium, pointing out the importance of alkaline conditions for O-
292
carboxymethylation. A sufficiently strong base is needed to allow chloroacetate
293
penetration on the whole TMC chain, avoiding side reactions between NaOH and
294
chloroacetate (Barros et al., 2013).
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Successful O-carboxymethylation of TMC is further demonstrated by
296
heteronuclear single quantum correlation (1H-13C HSQC) map (Fig. 5). The
13
297
signals that belong to the –COOH group substituted on OH-3 and OH-6 are present
298
at f1 =71.1 ppm and 71.5 ppm, respectively, and also appear on 1H-NMR spectra
299
(Fig. 4A) at 4.15-4.5 ppm (peak 8). Their positions on HSQC map indicate that there
300
are two possible sites for O-carboxymethylation on the TMC backbone, but the major
301
active site is on C-6 as shown by the presence of a peak at 4.15 ppm (Chen et al.,
302
2003; Chen & Park, 2003).
303 304 305
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C-NMR
Fig. 5. 1H-13C HSQC spectrum for CMTMC 2 with TSP as an internal standard. 3.1.2. Cytotoxicity
306
After 10 days of CMTMC suspension exposure to human dermal fibroblasts,
307
the results showed no significant toxicity (viability > 80% in all cases) (Fig. 6). The
308
lowest CMTMC concentration of 0.1 mg mL-1 resulted in highest cell viability (>
309
100%), while 1 and 0.5 mg mL-1 CMTMC showed a minor decrease in viability
13 Page 13 of 18
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(≈82%), as compared to the negative control (SDS 1%, 7 – 11% viability). This
311
absence of CMTMC cytotoxicity is consistent with literature reports (Yin et al., 2007).
Polymer concentration (mg mL-1)
312
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Cell viability (%)
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Fig. 6. Human dermal fibroblast viability upon exposure to CMTMC polymer solutions
314
for 0 to 10 days. Results are given as mean ± SD (n=3).
315
4.
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Conclusions
The present work demonstrates that O-carboxymethyl chitosan can be obtained
317
with unprecedented degree of carboxymethylation, higher than 85% based on O-
318
methylation free TMC. As for TMC synthesis, we determined optimal reaction time
319
and solvent ratio for precipitation. O-methylation-free TMC was obtained using the
320
Eschweiler-Clarke reaction step. O-Carboxymethylation was carried out using
321
sufficiently strong base in isopropanol. Further in vitro results indicated that CMTMC
322
was devoid of toxicity on human dermal fibroblasts with no negative influence on cell
323
growth and morphology.
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Taken together, these results provide a chitosan derivative platform for drug
325
delivery. The highly O-carboxymethylated chitosan provides a material for
326
subsequent derivatization with peptide, e.g., through carbodiimide coupling
327
chemistry, proteins or targeting moieties. The presence of charges enables
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formulation of nanoparticles, gels, or dried sponges as carriers, allowing multiple
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delivery modes for various biomedical applications.
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Acknowledgements We appreciate the financial support by Sciex-NMS grant 12.191. For the
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helpful discussions on NMR spectra we are grateful to Dr. Elisabeth Rivara-Minten
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(University of Geneva), Emmanuelle Sublet (University of Geneva) and Corinne
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Scaletta (CHUV, Lausanne) for their assistance during the cell culture experiments.
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Table 1.
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Increase of the degree of trimethylation during the reaction (n=3). Degree of quaternization (%)
100
24.5±1.2
140
36.8±9.3
overnight
22.7±3.3
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Reaction time (min)
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