Choline chloride-thiourea, a deep eutectic solvent for the production of chitin nanofibers

Article abstract of DOI:10.1016/j.carbpol.2013.12.082

Deep eutectic solvents (DESs) consisting of the mixtures of choline halide (chloride/bromide)-urea and choline chloride-thiourea were used as solvents to prepare α-chitin nanofibers (CNFs). CNFs of diameter 20-30 nm could be obtained using the DESs comprising of the mixture of choline chloride and thiourea (CCT 1:2); however, NFs could not be obtained using the DESs having urea (CCU 1:2) as hydrogen bond donor. The physicochemical properties of thus obtained NFs were compared with those obtained using a couple of imidazolium based ionic liquids namely, 1-butyl-3-methylimidazolium hydrogen sulphate [(Bmim)HSO₄] and 1-methylimidazolium hydrogen sulphate [(Hmim)HSO₄] as well as choline based bio-ILs namely, choline hydrogen sulphate [(Chol)HSO₄] and choline acrylate. The CNFs obtained using the DES as a solvent were used to prepare calcium alginate bio-nanocomposite gel beads having enhanced elasticity in comparison to Ca-alginate beads. The bio-nanocomposite gel beads thus obtained were used to study slow release of 5-fluorouracil, an anticancer drug.

Full text of DOI:10.1016/j.carbpol.2013.12.082

Accepted Manuscript
Title: Choline Chloride-thiourea, a deep eutectic solvent for
the production of chitin nanofibers
Author: Chandrakant Mukesh Dibyendu Mondal Mukesh
Sharma Kamalesh Prasad
PII:
S0144-8617(13)01297-6
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.carbpol.2013.12.082
CARP 8459
To appear in:
Received date:
Revised date:
Accepted date:
5-12-2013
25-12-2013
28-12-2013
Please cite this article as: Mukesh, C., Mondal, D., Sharma, M., & Prasad, K.,Choline
Chloride-thiourea, a deep eutectic solvent for the production of chitin nanofibers,
Carbohydrate Polymers (2014), http://dx.doi.org/10.1016/j.carbpol.2013.12.082
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High lights
1) Deep eutectic solvents were used for the production of chitin nanofibers.
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2) The physicochemical properties of the nanofibers thus obtained were compared with those obtained
using few ionic liquids.
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3) The nanofibers thus obtained were used to prepare calcium alginate bio‐nanocomposite gel beads.
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4) The nanocomposite gel beads were used as sustained drug release application for an anticancer drug, 5‐
fluorouracil.
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10 Choline Chloride-thiourea, a deep eutectic solvent for the production of
11 chitin nanofibers
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Chandrakant Mukesh, a Dibyendu Mondal, a Mukesh Sharma,a and Kamalesh Prasad a,b
*
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a AcSIR- Central Salt & Marine Chemicals Research Institute, G. B Marg, Bhavnagar-364002
(Gujarat), India
b Marine Biotechnology and Ecology Discipline, CSIR-Central Salt & Marine Chemicals
Research Institute, G. B Marg, Bhavnagar-364002 (Gujarat), India, * Corresponding author :
Tel : +91-278-2567760. Fax. +91-278-2567562 Email: kamlesh@csmcri.org /
drkamaleshp@gmail.com]
Abstract:
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Deep eutectic solvents (DESs) consisting of the mixtures of choline halide (chloride/bromide)-
urea and choline chloride-thiourea were used as solvents to prepare α-chitin nanofibers
(CNFs). CNFs of diameter 20-30 nm could be obtained using the DESs comprising of the
mixture of choline chloride and thiourea (CCT 1:2), however NFs could not be obtained using
the DESs having urea (CCU 1:2) as hydrogen bond donor. The physicochemical properties of
thus obtained NFs were compared with those obtained using a couple of imidazolium based
30 ionic liquids namely 1-butyl-3-methylimidazolium hydrogen sulphate [(Bmim)HSO4] and 1-
31 methylimidazolium hydrogen sulphate [(Hmim)HSO4] as well as choline based bio-ILs namely,
32 choline hydrogen sulphate [(Chol)HSO4] and choline acrylate. The CNFs obtained using the
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DES as a solvent were used to prepare calcium alginate bio-nanocomposite gel beads having
enhanced elasticity in comparison to Ca-alginate beads. The bio-nanocomposite gel beads
thus obtained were used to study slow release of 5-fluorouracil, an anticancer drug.
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1. Introduction
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Nanofibers (NFs) are extensively used in nanotechnology for the preparation of functional
nanocomposites (Huang et al., 2003). The widely used technique to produce NFs is the
electrospinning process, which uses an electrical charge to draw very fine fibers from polymer
solutions (Subbiah et al., 2005). NFs are also useful in various biomedical applications and
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hence substantial efforts are being made to develop biodegradable polymer scaffolds suitable
for tissue engineering applications (How et al., 1992 ; Vacanti & Vacanti, 1997). Matthews et
al., (2002) have attempted to prepare tissue-engineering scaffolds composed of collagen
nanofibers by optimising various parameters of electrospinning process (Matthews et al.,
45 2002). Cellulose NFs were prepared directly from wood by Abe et al., (2007).
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Chitin, the (1-4)-2-acetamido-2-deoxy-β-D-glucan, is industrially produced from marine
resources (Muzzarelli, 2012; Muzzarelli et al., 2012). Chitin and chitosan activate the
macrophages, and are mucoadhesive, antimicrobial, biodegradable and nontoxic and
therefore they are widely used for the repair of wounded human tissues (Muzzarelli, 2009).
Chitin has been processed in the form of nanofibers (CNFs) or nanocrystals (CNC) by several
research groups by employing various techniques, such as acid hydrolysis (Morin and
Dufresne, 2002; Lu, Weng and Zhang, 2004; Goodrich and Winter, 2007), TEMPO mediated
oxidation (Fan, Saito and Isogai, 2008a ; Fan, Saito and Isogai, 2008b), ultrasonication (Zhao,
54 Feng and Gao, 2007) and electrospinning (Jayakumar et al., 2010). More recently, Isogai et
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al., (2012) have reported comparative characterization of aqueous dispersions and cast films
of different chitin nano whiskers (Fan, Fukuzumi, Saito et al., 2012). Nanofibrillation efficiency
of α-chitin in various organic and inorganic acids by varying the pH and ionic strength was
58 reported (Qi et al., 2013). CNFs having good dispersibility in 2,2,2-trifluoro ethanol and
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preparation of its nanocomposite with polycaprolactone is reported (Ji et al., 2012).
Ionic liquids (ILs) are low melting point salts that form liquids at temperatures below the
boiling point of water. Applications of ILs in carbohydrate and polysaccharide chemistry are
increasing and they are being used to prepare a number of new compounds (El. Seoud, et al.,
2007). Biomedical exploitation of chitin is currently under way with the aid of ionic liquids
(Muzzarelli, 2011). Attempts are also made to process chitin in NF form using ILs e.g., 1-
ethyl-3-methylimidazolium acetate was used to dissolve crustacean shells and highly pure high
molecular weight chitin powder as well as fibers was recovered (Qin et al., 2010; Barber et al.,
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2013). Kadokawa et al., (2011) have demonstrated application of 1-ethyl-3-methylimidazolium
bromide for the preparation of chitin nanowhiskers. Deep eutectic solvents (DES) are fluids
obtained by heating two or more compounds capable of self-association through hydrogen
bond interactions and have lower melting points in comparison to each of the individual
components (Zhang et al., 2012). Abbott et al., (2004) for the first time presented DESs as
suitable alternative solvents to ILs. DES has similar physicochemical properties in comparison
to ILs but they are considered different from ILs mainly because (a) they do not entirely consist
of ionic species and (b) they can be prepared from non-ionic species as well unlike the ILs
(Zhao and Qu, 2013). Further, the DESs are more advantageous due to their cheaper cost
and environmentally friendlier nature. Owing to these remarkable advantages, DESs are now
of growing interest in many fields of research e.g., catalysis, organic syntheses, dissolution
media, extraction processes, electrochemistry and material chemistry (Zhang et al., 2012). We
have demonstrated recently suitability of few bio-ILs and DESs for the solubilisation of natural
polymers such as DNA and α-chitin (Mukesh et al., 2013; Mondal et al., 2013; Sharma et al.,
2013).
Herewith, we report production of chitin nanofibers using a deep eutectic solvent
(choline chloride-thiourea) and application of the nanofibers as reinforcement fillers for calcium
alginate beads having ability to release 5-Fluorourascil, an anticancer drug. The
85 physicochemical properties of the NFs further compared with those prepared using a couple of
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imidazolium based ionic liquids namely 1-butyl-3-methylimidazolium hydrogen sulphate
(BmimHSO4) and 1-methylimidazolium hydrogen sulphate (HmimHSO4) as well as choline
based bio-ILs namely, choline hydrogen sulphate (Chol.HSO4) and choline acrylate.
2. Materials and methods
2.1. Materials
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Choline chloride, 1-methylimidazole, 5-fluorouracil and chitin powder obtained from crab shells
were purchased from TCI Fine chemicals, Tokyo, Japan. The degree of polymerization of
chitin from the origins was reported to be ca. 2000–4000 (Hasegawa et al., 1994; Kurita,
2001). The degree of acetylation of the chitin sample was estimated by elemental analyses
data and found to be 94.1%, which was in good agreement with that of standard chitin
(Guinesi and Cavalheiro, 2006). The FT-IR spectra of the sample showed vibrational bands at
1662 cm-1 and 1631 cm-1 in the amide I region characteristic of α-chitin (Cárdenas et al., 2004).
The band at 1540 cm-1 corresponds to protein absorption, which are absent in FT-IR spectrum
indicating absence of protein impurities in the sample. 1-Butyl-3-methylimidazolium chloride
was purchased from Merck & Co., Germany. Sodium alginate was purchased from Sigma-
Aldrich Chemical Co., USA. Thiourea, acrylic acid (AA), and CaCl2 were purchased from S.D.
Fine chemicals, Mumbai, India. All chemicals were of analytical grade and were used as
received without further purification.
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2.2. Measurements
Powder X-ray diffraction patterns were recorded at 298 K on a Phillips X'pert MPD system
using CuKα radiation (λ = 0.15405 nm) with 2θ range from 10 ° to 80 ° at a scan speed of 0.1
°sec−1. The dry samples were placed on carbon coated copper grids (300 mesh sizes) and the
transmission electron microscopic (TEM) images of thus prepared samples were obtained
using a JEOL transmission electron microscope (Model JEM 2100, Japan) operated at
accelerating voltage of 120 kV. The dry samples were dispersed in acetone and coated on
aluminum stubs and evaporated to dryness followed by recording of their SEM images on a
LEO 1430 VP instrument employing accelerating voltage of 18 kV. FT-IR analyses were
carried out on a Perkin Elmer Spectrum GX, FTIR System, USA by taking 2.0 mg of sample in
115 600 mg of KBr. All spectra were averages of two counts with 10 scans at a resolution of 5 cm-
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1. H NMR of the ILs and DES were recorded on a Bruker Avance-II, 500 MHz spectrometer.
Linear viscoelastic properties (controlled deformation mode with 0.01% strain of Ca-Alg beads
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and Ca-Alg/CNF bio-nanocomposite beads were carried out on an Anton Paar, Physica MCR
301 rheometer USA, using parallel plate PP50/P-PTD200 geometry (50 mm diameter; 0.1 mm
gap) operating in dynamic mode. All the dynamic rheological data were checked as a function
of strain amplitude to ensure that the measurements were performed in the linear viscoelastic
region. The UV-Vis absorption spectra of acidic ionic liquids were recorded on a Varian CARY
500 UV-Vis-NIR Spectrophotometer, USA. CNF solutions in distilled water were used for AFM
sample preparation. Appropriately diluted nanofibers (0.05 mg/mL) solutions were deposited
on freshly cleaved mica foil. After 2 min the solution was drained off and dried by nitrogen gas.
The mica foil was then kept in dust free CaCl2 desiccators for five days and then was used for
AFM analysis. The AFM measurements were performed in the semi contact mode using an
Ntegra Aura (Nt-Mdt, Russia) instrument at room temperature in air. The height of images was
recorded from different area of each sample. The software used for image analysis was
‘‘Nova’’. Elemental analyses were carried out on a Perkin-Elmer CHNS analyser.
2.3. Synthesis of DESs and acidic ionic liquids
In a typical reaction for the synthesis of 1-butyl-3-methylimidazolium hydrogen sulphate
133 [(Bmim)HSO4], [(Bmim)]Cl (5 g, 0.0286 mol) was added into 20 ml dichloromethane in a
134 round-bottom flask followed by drop wise addition of one equivalent of sulphuric acid (98%)
135 (1.5 ml, 0.0286 mol) for 10 min at room temperature. The reaction mixture was refluxed for 24
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h at 70 °C and the resulting IL was separated out, washed with ethyl acetate two times and
dried at 70 °C for 6 h under vacuum. Similar metathesis reaction was carried out for the
synthesis of the other two ILs viz., choline hydrogen sulphate [(Chol)HSO4], methyl
139 imidazolium hydrogen sulphate [(Hmim)HSO4] and choline acrylate. The structures were
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confirmed by 1H NMR and mass spectrometry (ESI-MS).
DESs were prepared following the method described by Abbot et al. (2004). In a typical
reaction, both the hydrogen bond donor (HBD) and acceptor (HBA) molecules were heated at
50 oC (reactions where urea was used as HBD) and 70 oC (reactions where thiourea was used
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as HBD) with constant stirring at optimized mole ratios as shown in Table 1 under argon
atmosphere until homogenous and colorless liquids were formed (Supporting Figure S1).
<Table 1>
2.4. Preparation of α-chitin nanofibers
148 Pure chitin powder (at optimized concentration of 10% w/w) was dispersed separately in DESs
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and acidic ILs followed by stirring at 500 rpm at 100 oC for different duration of time as shown
in Table 1. The gel like materials thus obtained was diluted by adding 10 mL of distilled water.
The dispersed chitin particles (Figure 1) were collected after centrifugation at 10000 rpm for 10
minutes followed by washing with distilled water to make them free from acid (Step 1). The
153 acid free chitin was further dispersed in distilled water (30 ml) and the solution was ultra
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sonicated using an ultra sonication rod (35 amplitude, 0.5 cycle, 40 minutes). The NFs were
isolated from this solution by lyophilization (Step 2). The water present in the ionic liquid/DES
and water mixture obtained in step 1 was evaporated using a rotor vapor to obtain the recycled
ILs/DESs. The purity of thus obtained solvents was checked by 1H NMR.
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2.5. Preparation of Ca-Alginate/chitin nanofibers bio-nanocomposite (Ca-Alg/CNF)
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Homogeneous mixture of 1% w/v of Na-alginate and 0.4% CNF in deionized water was taken
in a surgical syringe attached to a needle. The mixture was then poured drop wise into 5%
161 CaCl2 aqueous solution under stirring. The Ca-alginate beads thus formed were isolated from
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the solution by filtration and washed several times with distilled water to remove adhered salts
on the bead surfaces.
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2.6 Release of 5-fluorouracil (5-UF) from Na-Alg/CNF gel beads
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Homogeneous mixture of 1% w/v of Na-alginate, 0.4% CNF and 0.5 mg/mL of 5-UF in
deionized water was taken in a surgical syringe attached to a needle. The mixture was then
poured drop wise into 5% CaCl2 aqueous solution containing 0.5 mg/mL of 5-FU under stirring
(Supporting Figure S2). The beads were lyophilised and known amounts of the thus obtained
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beads (500 mg) were accurately weighed and added to 25 ml of pH 7.4 phosphate buffer
(PBS). The sample was placed in an ultrasonic bath (Elma, Germany) for 10 min. to ensure
maximum extraction of encapsulated 5-FU in the buffer. The sample was then made up to 10
mL using pH 7.4 phosphate buffer. An aliquot sample was assayed at 266 nm using an UV-
spectrophotometer.
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3. Results and Discussion
We have recently observed that DES consisting of the mixture of choline chloride and
urea or thiourea can solubilize chitin between 5-9% w/w (Sharma et al., 2013). 100 to 600 mg
of chitin (2-12% w/w) was added into vials containing 5 g of solvent (separately for DES and
ILs) placed in an oil bath with vigorous stirring at 100 oC for different duration of time (Table 1).
179 It was observed that, chitin with concentrations up to 8% w/w produced dilute solutions making
180 the isolation of the nanofibers very difficult. Upon dissolution, 9% w/w chitin gave formation of
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a viscous solution, while 10% w/w chitin produced soft gel which could be processed to the
182 form of nanofibers following method shown in Figure 1. However, the concentration of chitin
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higher that 10% w/w yielded relatively stronger gel and nanofibers having enhanced length
and diameter (outside nano meter range) could be isolated. Considering these, chitin with 10%
w/w was considered as optimized concentration for the preparation of the CNFs. After the
isolation of the nanofibers the corresponding DES or IL could be recycled with reasonably
good yield (90-92%) and was reused in the NF preparation process. It was observed that, the
recovery of DES was bit lesser in comparison to the recovery of the ILs. It should be noted
that, the DESs comprising of choline chloride /bromide and urea did not result formation of
nano fibers. The isolated chitin from these solutions showed presence of agglomerated
structures (supporting Figure S3) rather than fibers. On the other hand, chitin isolated from
choline chloride-thiourea 1:2 (CCT 1:2) showed formation of nanofibers under SEM
(supporting Figure S4). The morphology of chitin before treatment showed presence of thicker
fibers (supporting Figure S5). CNFs also could be isolated from all the other ILs except choline
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acrylate used in the investigation. Choline acrylate gave formation of fibers with agglomerated
morphology, perhaps due to the thermal polymerization of the IL. Since the acid hydrolysis
was the prime reason behind formation of CNFs, the acidity of the ILs as assessed using
Hammett functions was responsible for the formation of the CNFs (Table S1). Further, the acid
character of DESs perhaps helped the formation of the CNFs. The yield of CFNs prepared in
CCT 1:2 was 84% w/w, while the highest yield of 93% w/w was observed for the NFs prepared
201 in (Bmim) HSO4 followed by (chol)HSO4 (86%) and (Hmim)HSO4 (77%) [Table 1].
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<Figure 1>
The FT-IR spectra of the CNF prepared using CCT 1:2, (Bmim)HSO4, (Hmim)HSO4
and (chol)HSO4 showed vibration bands at 1663 and 1631 cm-1 characteristic of α-chitin
205 (amide-I region), similar to the pure α-chitin, which indicates preservation of backbone
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structure of chitin in the NFs (Supporting Figure S6). Powder XRD pattern of the CNFs
prepared in the DES and the ILs (Supporting Figure S7) mainly showed four diffraction peaks
208 at around 9.5 o, 19.5 o, 20.9 o, and 23.4 o, which were assigned to 020, 110, 120, and 130
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planes of chitin and corresponds to the crystalline structure of α- chitin and is in good
agreement that with chitin powder (Supporting Figure S7a). These data indicated that the
211 crystalline structure of α-chitin remained intact during the preparation of the NFs and their
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reconstruction from the solutions (Kadokawa et al., 2011). 13C-solid NMR (SSNMR) showed
presence of characteristic carbonyl band of the amide (δ176 ppm) for chitin in all the CNFs
prepared in the above solvent systems. The representative spectra are shown in Supporting
Figure S8. All the above results confirmed the preservation of structural integrity of the
biopolymer in the NFs.
The TEM images of the regenerated NFs from CCT 1:2 showed formation of NFs of
diameter 20 ± 6 nm having length 1 µm ± 0.5 µm (Figure 2a) (average of fifteen NFs). On the
other hand as evident from the images (Figure 2b-d) the NFs prepared in the ILs had a bit
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higher diameter and lesser length in comparison to the NFs prepared in the DES [e.g.,
diameter and length of 28 ± 6 nm and 1.5 ± 0.5 µm for NFs isolated from (Bmim)HSO4.
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The AFM images of the CNFs prepared from CCT 1:2 showed formation of fibers of
width 25-45 nm and length 162-450 nm. The height of the fibers was ca. 40 nm. On the other
224 hand the CNF prepared in (Hmim)HSO4 had marginally higher width (30-40 nm) and
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substantially lower length (110-250 nm) in comparison to the NFs prepared in the above DES
(Figure 3). The NFs prepared in the rest of the ILs also showed the similar trend.
After the isolation of the NFs, the corresponding DESs or ILs were recycled and the
purity of the recycled solvents was found to be equivalent to that with respective unprocessed
solvents. The recycled solvents thus obtained were reused for the preparation of the CNFs
following the same scheme shown in Figure1. The SEM and TEM images of the fibers showed
formation of NFs of similar dimensions as obtained for the pure solvents proving the efficiency
of the recycled solvents for reuse in the process (Supporting Figure S9 & S10).
The calcium alginate beads embedded with the CNFs prepared in the DES were
prepared as described above. The phase contrast optical micrographs (100 X) of the cross
section of the beads showed presence of clusters of the NFs (Supporting Figure S11). The FT-
IR spectra of the bio-nanocomposite gel beads showed presence of CNFs in the beads
(Supporting Figure S12) evident from the appearance of the characteristic 1640 cm-1 band of
the CNFs in bio-nanocomposite gel beads confirmed formation of the bio-nanocomposite. The
elemental analyses of gel beads showed presence 65.5% w/w of CNFs in the gel bio-
nanocomposite beads.
Monitoring of the G´G´´ vs time of the 1% w/w Ca-Alg gel bead and Ca-Alg / CNF bio-
nanocomposite gel beads showed a three fold enhanced values of both the moduli for the bio-
nanocomposite beads in comparison to the Ca-Alg beads indicating higher elasticity for the
composite gel beads resulted from the enforcement by the CNFs (Supporting Figure S13).
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Alginates have been widely used in pharmaceutical industry for controlled drug release.
Arica et al., (2002) have used Ca-Alg beads as a drug slow release matrices for 5-FU and
studied effect of polymer concentration and the drug loading on the release profile of the drug
and observed about 80% release of the drug (Arica et al., 2002). To study the slow release of
5-FU from the Ca-Alg/CFN bio-nanocomposite and Ca-Alg gel beads, weighed amount of 5-
FU loaded samples were put into a glass vessel containing 25 ml of phosphate buffer solution
(pH 7.4) at 37±0.5 °C. At scheduled time intervals, 1 ml of sample was removed from the
vessel and the amount of 5-FU released from the matrices was determined by UV-Vis
spectrophotometer at 266 nm upto 24 h. It was observed that, bio-nanocomposite gel beads
were able to release about 70% of the drug after 24 h at this pH, whereas the control Ca-Alg
beads rapidly released 39% of drug after 3 h as shown in Figure 4. The higher release rate
and extended duration of release characteristics of the composite gel beads indicated the
crucial role of the CNFs in the sustained release of the drug at pH 7.4 (pH of the colon).
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4. Conclusions
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α-Chitin nanofibers were prepared by solubilizing chitin in deep eutectic solvents
comprising of the mixture of choline chloride and urea or thiourea as well as in a couple of
imidazolium based and choline based bio-ionic liquids. DES comprising the mixture of choline
chloride and thiourea yielded nanofibers, while the mixture with urea did not produce
nanofibers. Formation of chitin nanofibers in all the acidic ionic liquids except choline acrylate
was observed. The results demonstrated the suitability of the DESs for the preparation of
chitin nanofibers. The efficiency of the nanofibers as enforcement filler for calcium alginate gel
beads was also demonstrated by preparing chitin nanofiber embedded calcium alginate bio-
nanocomposite gel beads with enhanced elasticity and ability to release 5-fluorouracil, an anti
cancer drug at pH 7.4 (pH of the colon).
Acknowledgement: KP thanks Council of Scientific & Industrial Research, New Delhi for
granting CSIR-Young Scientist Awardees Project and overall financial support. CM, MS and
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DM thanks UGC and CSIR for Junior Research Fellowships. Department of analytical science
& centralized instrumentation facility of the institute is gratefully acknowledged for providing all
necessary analytical facilities for the work.
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Figure captions
401
402
403
404
405
406
407
408
Figure 1. Photographic demonstration for the preparation of α-chitin nanofibers and recycling
of ionic liquid or deep eutectic solvents.
Figure 2. Transmission micrographs of α-chitin nanofibers prepared using (a) choline
chloride-thiourea 1:2 (b) (Bmim)HSO4 (c) (Hmim)HSO4 (d) (Chol) HSO4
Figure 3. Atomic force micrographs of α-chitin nanofibers prepared using (a) choline chloride-
thiourea 1:2 and (b) (Hmim)HSO4
409
410
411 Figure 4 . In vitro release profile of (a) 5-FU loaded Ca- alginate/chitin nanofibers bio-
412
nanocomposite gel beads and (b) calcium alginate beads.
413
414
415
Table 1: Preparation of chitin nanofibers in deep eutectic solvents and ionic liquids (chitin
concentration with respect to solvent was 10% w/w and temperature of reaction was 100 oC)
Deep Eutectic Solvents
Optimized molar Formation Time
Yield of nanofibers
wt%
ratio (HBA :
HBD)
of
(h)
nanofibers
Hydrogen
Bond
Hydroge
Bond
DES
ILs
n
acceptors
(HBA)
Donors
(HBD)
Choline
Chloride
Urea
1:2
1:2
No
2
2
2
NA
NA
84
--
Choline
Bromide
Urea
No
----
---
Choline
Chloride
Thiourea 1:2
Ionic liquids
Yes
(Chol)HSO4
Yes
Yes
Yes
No
1
1
1
2
---
---
---
---
86
77
93
….
(Hmim)HSO4
(Bmim)HSO4
Choline Acrylate
416
417
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418
419
420
421
422
Figure 1. Photographic demonstration for the preparation of α-chitin nanofibers and recycling of ionic
liquid or deep eutectic solvents.
423
424
425
426
427
428
Figure 2. Transmission micrographs of α-chitin nanofibers prepared using (a) choline chloride-
thiourea 1:2 (b) (Bmim)HSO4 (c) (Hmim)HSO4 (d) (Chol) HSO4
16
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429
430
Figure 3. Atomic force micrographs of α-chitin nanofibers prepared using (a) choline chloride-thiourea
431 1:2 and (b) (Hmim)HSO4
432
433
434
435
436
437
438
439
440
441
442
443
444
445
446
447
448
Figure 4. In vitro release profile of (a) 5-FU loaded Ca- alginate/chitin nanofibers bio-nanocomposite
gel beads and (b) calcium alginate beads.
449
450
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