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wheat cellulose scaffold
Accepted Manuscript Title: IN-VITRO RELEASE OF FRAGRANT L-CARVONE FROM ELECTROSPUN POLY ( − CAPROLACTONE)/WHEATCELLULOSESCAFFOLD Author: Ramamoorthy Manjula Sheeja Rajiv PII: DOI: Reference:
S0144-8617(15)00644-X http://dx.doi.org/doi:10.1016/j.carbpol.2015.07.015 CARP 10117
To appear in: Received date: Revised date: Accepted date:
3-3-2015 17-6-2015 2-7-2015
Please cite this article as: Manjula, Ramamoorthy., & Rajiv, Sheeja., IN-VITRO RELEASE OF FRAGRANT L-CARVONE FROM ELECTROSPUN POLY ( − CAPROLACTONE)/WHEATCELLULOSESCAFFOLD.CarbohydratePolymershttp : //dx.doi.org/10.1016/j.carbpol.2015.07.015 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.
[1]
1
IN-VITRO RELEASE OF FRAGRANT L-CARVONE FROM ELECTROSPUN POLY (ɛ-
2
CAPROLACTONE) / WHEAT CELLULOSE SCAFFOLD
3
Ramamoorthy Manjula and Sheeja Rajiv*
4
Department of Chemistry, Anna University, Chennai, Tamilnadu 600 025, India.
5
*Corresponding author: Dr.Sheeja Rajiv
6
E-mail: [email protected] ; Tel: +914422358658, Fax: +914422200889
7 8 9
HIGHLIGHTS
10 11
L-Carvone loaded PCL /Wheat cellulose (WC) nanofibers were prepared by the electrospinning technique which showed good antimicrobial activity.
The in-vitro release of L-Carvone from the PCL-WC fibers was found to follow
12
Korsmeyer Peppas kinetic model which indicated diffusion controlled mechanism of
13
release.
14 15 16
Bioactive PCL-WC scaffold is an ideal fragrant scaffold for antimicrobial textile applications.
ABSTRACT
17
The release kinetics of L-Carvone loaded from electrospun Poly (ɛ-caprolactone)
18
(PCL) and Wheat cellulose (WC) blend were studied. WC was extracted from wheat straw, a
19
cost effective agricultural waste by the acid hydrolysis method. A homogeneous solution of
20
PCL-WC (13:3 wt%) was optimised to produce beadless electrospun PCL-WC blend
21
nanofibers. Further, WC and the prepared electrospun PCL-WC blend fibers were
22
systematically characterised by ATR-FTIR,
23
measurements. The hydrophilic character of the blend fibers was analysed using swelling
24
tests and contact angle measurements. The loading efficiency of L-Carvone into the
25
electrospun PCL-WC blend fibers was evaluated to be ~ 70%. The in-vitro release of L-
26
Carvone from PCL-WC blend fibers followed Korsmeyer Peppas kinetic model indicating
SEM, XRD, TGA, DTGA, and DSC
[2]
27
the diffusion mechanism and the maximum release of L-Carvone was found to be ~ 84%
28
over a period of 30 h. These results would offer the prepared PCL-WC blend as an ideal
29
fibrous mesh for fragrant antimicrobial textile applications.
30 31
KEY WORDS: L- Carvone; Wheat Cellulose; Blend; Poly (ɛ-caprolactone); Release kinetics;
32
Korsmeyer Peppas model
33 34 35
1.0
INTRODUCTION
36
Over the past few decades, electrospun biocomposite nanofibers are of great
37
importance, owing to their unique properties like high porosity, large surface area, small pore
38
size, superior mechanical and thermal properties (Miao et al., 2012).
39
cellulose-based nanofibers, nanoparticles, nanocrystals are reported to provide many
40
advantages comprising world wide availability from various non-conventional resources, low
41
cost, better biodegradability and easy tailor made process ability (Azizi Samir, Alloin, &
42
Dufresne, 2005; Hou, Zhou, & Wang, 2009). The use of natural cellulose fibers consist of a
43
major research work in the production of biodegradable composites. The reinforcement of
44
cellulose nanoparticles has attracted significant attention due to their abundance, renewable
45
nature, large surface area and good mechanical strength (Chazeau, Cavaille, Canova,
46
Dendievel, & Boutherin, 1999; Choi, & Simonsen, 2006; Helbert, Cavaille, & Dufresne,
47
1996; Wang, Sain, & Oksman, 2007).
Cellulose and
48
Essential oils have excellent antibacterial, antifungal, antioxidant, insect-repellent and
49
insecticidal properties (Bilia, Guccione, Isacchi, Righeschi, Firenzuoli, & Bergonzi, 2014;
50
Fukumoto, Sawasaki, Okuyama, Miyake, & Yokogoshi, 2006). However, the highly volatile
51
nature and degradation upon exposure to sunlight limits their successful application. Recent
[3]
52
researchers made several formulations in order to achieve chemical stability, a controlled
53
release, increased efficiency and activity of essential oil. To ensure safer and easier
54
handling of essential oils, they are entrapped into solid carriers which retain the bioactive
55
compounds and further enables controlled release of the ingredients thereby enhancing their
56
bioavailability and efficacy (Ortan, Ferdes, Rodino, Pirvu, &
57
biodegradable and biocompatible systems have been reported for the encapsulation and
58
sustained release of essential oils (Dimaa, Gitina, Alexea, & Dimab, 2013; Ghosh,
59
Mukherjee, & Chandrasekaran, 2013; Ortan, Campeanu, Dinu–Pirvu, & Popescu, 2009;
60
Sanna passion, Bazzoni, & Moretti, 2004; Shirwaikar, Prabhu, & Kumar, 2008; Soliman, El-
61
Moghazy, El-Din, & Massoud,. 2013). PCL is one of the excellent biodegradable and semi
62
crystalline linear hydrophobic polymers, which find many applications in biomedical field due
63
to their good biocompatibility, superior mechanical properties and complete degradation to
64
non-toxic by-products (Zhang, Gupte, & Ma, 2013). Moreover, PCL nanofiber matrices
65
have slower degradation rates among the well-known biodegradable synthetic polyesters
66
such as PGA, PLGA and PLA due to the presence of five hydrophobic –CH2 moieties in the
67
repeating units (Hao, Yuan, & Deng, 2002; Kanani, & Bahrami, 2011). The main aim of the
68
present study was to fabricate the L-Carvone loaded PCL-WC electrospun blend fibers using
69
electrospinning method. The cellulose extracted from wheat straw fibers were added to PCL
70
to improve the biodegradability and wettability of PCL thereby making the scaffold suitable
71
as an antimicrobial fragrant scaffold. A homogeneous solution of PCL-WC was prepared and
72
electrospun to obtain PCL-WC blend fibers. Further, the L-Carvone was blended with the
73
PCL-WC solution and electrospun to form the L-Carvone loaded PCL-WC blend fibers. The
74
kinetics of the ‘in-vitro’ release of L-Carvone from the blend fibers was fitted with four
75
different kinetic models. In addition, the essential oil was highly active even after the release
76
from the electrospun fibers which were confirmed by their antimicrobial activity against gram-
77
positive and gram-negative microorganisms.
78 79
2.0
MATERIALS AND EXPERIMENTAL METHODS
Draganescu, 2013). Many
[4]
80
2.1
MATERIALS
81
Wheat straw used in the present work was received from Dharmapuri, Tamil Nadu,
82
India. PCL (Mn ~ 70000 – 90000) and L-Carvone ( 97%) were obtained from Sigma-Aldrich,
83
India. Chloroform, Trifluoroacetic acid (TFA), Ethanol, Sodium hydroxide (NaOH), Sulphuric
84
acid (H2SO4), Hydrogen peroxide (H2O2), Nitric acid (HNO3) were purchased from Fischer
85
Scientific Company. All the reagents used were of analytical grade.
86 87 88 89
2.2
EXTRACTION OF CELLULOSE FROM WHEAT STRAW
90
Initially, wheat straw was washed with distilled water to remove impurities
91
covering the external cell wall. The baled wheat straw was cut manually to 1-2 cm. The
92
cut wheat straw was pre-treated
93
agitation to remove lignin (Alemdar & Sain 2008). The extraction of wheat cellulose (WC)
94
was carried out according to the procedure reported for extraction of cellulose from sisal
95
fibers (Moran, Alvarez, Cyras, & Vazquez, 2008).
with 3% NaOH at 50oC for 2 h under continuous
96 97
2.3
FABRICATION OF ELECTROSPUN PCL-WC BLEND FIBRES
98
The homogeneous solutions of 13 wt% PCL and 3 wt% WC were prepared using
99
chloroform and TFA solvents respectively. Then, PCL-WC blend solution was prepared by
100
mixing the solution of WC to the PCL solution over a period of 30 min until the solution
101
became homogeneous. The polymer blend solution was taken in a 5 ml syringe having a
102
needle tip of 0.6 mm inner diameter. A high voltage of 25 kV was supplied directly from a
103
high DC voltage power supply to the needle and the negative terminal of the power was
104
connected to the collector covering with an aluminium foil. The polymer solutions were
105
electrospun at a flow rate of 0.9 ml/h and the tip-to-collector distance of 20 cm.
106
[5]
107
2.4
PREPARATION OF L-CARVONE LOADED PCL-WC BLEND FIBERS
108
A known weight of sample of standard size (1 x 1 cm) of PCL-WC blend nanofibrous
109
membrane was immersed in a 5 wt% of L-Carvone and allowed to absorb the active agent
110
over a period of 24 h at room temperature of 28oC (Peppas, & Am Ende, 1997).
111 112
2.5 MEASUREMENTS AND CHARACTERISATIONS
113
The size distribution of the extracted wheat cellulose was studied using a Malvern Zeta Sizer
114
Nano-S Version 7.03. The analysis was carried out using water as dispersant at 25oC in
115
order to measure the particle size. Approximately, 250 measurements were taken to
116
measure
117
Spectrophotometer equipped with a diamond crystal at an angle of incidence of 180°) was
118
used to identify the functional groups present in the WC and electrospun PCL-WC blend
119
fibers in the range of 400 to 4000 cm-1. The morphological features of wheat straw, extracted
120
WC and the prepared electrospun nanofibers were studied using Scanning Electron
121
Microscopy (FEI Quanta FEG 200 HRSEM). The samples were sputter coated with gold
122
under a fine coater for 120 s. An accelerating voltage of 10 kV was applied to observe SEM
123
images and the diameters of the isolated nanofibrils were measured using the Adobe
124
Photoshop CS3 Extended Software PS version 10.0 model. X-ray diffraction patterns of the
125
untreated straw, obtained WC and all prepared nanofibers was performed using a X’pert Pro
126
PANalytical Instrument using Cu Kᾳ radiation (λ=1.5418 Ao) in the 2 scale from 5-60o. The
127
thermal degradation characteristics of all samples were examined using thermogravimetric
128
analysis (TGA/DTA Model SDT 2600) at a heating rate of 10oC/min from 35 to 800oC with
129
continuous nitrogen flow of 20 cm3/min. The DSC measurements were performed with all
130
samples under nitrogen atmosphere with a scanning speed of 10oC/min and a heating rate
131
of 10oC/min from 0 to 750oC using DSC Q200 V24.4 Build 116 Model. In addition, the
132
degree of crystallinity (Xc) of PCL-WC nanofibers was calculated by dividing the measured
133
enthalpy of fusion (Hf) from DSC thermogram with the standard enthalpy of fusion for the
the
size
distribution of
the
sample.
ATR-FTIR analysis (Perkin-Elmer
[6]
134
100% crystalline PCL polymer (Hf0 = 139.5 Jg-1) (Crescenzi, Mancini, Calzolari, & Borri,
135
1972). The swelling characteristics of the fibers in deionised water were studied using the
136
reported procedure (Elayaraja, et al., 2011). In addition, the hydrophilicity of the prepared
137
fibers were studied by the contact angle measurements using an Euromex Optical
138
Microscope equipped with a CCD camera (Thangaraju, Srinivasan, Kumar, Sehgal, & Rajiv,
139
2012).
140 141 142 143 144
2.6
DETERMINATION OF LOADING EFFICIENCY OF L-CARVONE
145
The actual quantity of L-Carvone in the PCL-WC fibers was determined using UV-
146
Visible spectrophotometer at 236 nm. The amount of L-Carvone loaded in the blend fibres
147
was determined according to equation (1). All the experiments were performed in triplicates.
148
149
(%) =
× 100
(1)
150 151
2.7
IN-VITRO RELEASE STUDIES OF L-CARVONE AND KINETIC MODELLING
152
A known weight of L-Carvone loaded PCL-WC fibrous mat was placed in a 20 ml vial
153
containing absolute ethanol at 28oC. At regular time intervals, 5 ml of the aliquot was taken
154
and replaced with the same quantity of ethanol. The amount of L-Carvone in the releasing
155
medium was determined using UV-Visible spectrophotometer at the absorption wavelength
156
of 236 nm. The in-vitro release of L-Carvone was fitted into zero order, first order, Higuchi
157
and Korsmeyer peppas equations respectively to study the kinetic mechanism of L-Carvone
158
release from the fibers ( Sahoo, Chakraborti, & Behera, 2012; Zhao et al. 2012).
159
The four different kinetic model equations are as follows:
[7]
Q = Q -
160
Zero order equation :
161
First order equation:
162
Higuchi equation:
163
Korsmeyer – Peppas equation:
164
Where,
165
time t,
166
2.8
,
t
ln = ln Q Q =
(2) t
(3)
/
(4) /
=
(5)
are the release rate constants,
/
is fraction of oil released at
is constant and n is the diffusion constant that represents the release mechanism. ANTIBACTERIAL ACTIVITY OF THE L-CARVONE LOADED PCL-WC FIBERS
167
The antibacterial activities of the PCL-WC scaffolds were studied using gram positive
168
and gram negative, Staphylococcus aureus and Escherichia coli respectively as test
169
pathogens by Kirby- Bauer disk diffusion method. These bacterial pathogens were spread on
170
the L-Carvone loaded fibers in nutrient agar test plates under sterile conditions and
171
incubated for a period of 24 h and the zone of inhibition was measured.
172
3.0
RESULTS AND DISCUSSION
173
3.1
CELLULOSE YIELD
174
The wheat straw raw material was subjected to a delignification process in order to
175
facilitate the removal of lignin (Alemdar & Sain 2008). Initially, the crushed wheat straw fibers
176
were treated with alkali to remove the wax and partial separation of the cellulose fibers from
177
the cell wall. The second treatment with sulphuric acid led to more removal of non-
178
cellulosic substances and
179
treatment with nitric acid increased the crystallinity and molecular weight of cellulose
180
fibers resulting in a good yield and better quality of WC product in the nano scale as
181
reported (Moran et al., 2008).
182
3.2
formed
highly
stable
cellulose fibers. Moreover, further
PARTICLE SIZE DISTRIBUTION OF THE EXTRACTED WC
183
The acid treatment provided a viable and simple method for synthesising
184
nanocellulose. The average particle size of the extracted WC after the chemical treatment
[8]
185
and their average size distributions are shown in Fig. 1. The acid treatment of wheat straw
186
yielded fibrils with a Z-average diameter of 1.0 μm. The average particle size of the
187
extracted cellulose fibrils were found in the diameter range of 712-825 nm. As can be seen
188
from the Fig. 1, WCs were found to be in the diameter range of 825-955 nm with 33.6% of
189
the total intensity. Almost 53.7% of the particles were found to be in the diameters of
190
approximately 712-825 nm and only 12.7% of particles were found to have diameters in the
191
range of 600-712 nm.
192 193
Fig. 1. Particle Size distribution of extracted WC
194 195
3.3
MORPHOLOGY ANALYSIS
196
The SEM images of untreated wheat straw, WC and electrospun blend fibers are
197
shown in Fig. 2(a) – (e). SEM analysis is one of the very familiar method to examine the
198
surface morphology of the extracted cellulose and the electrospun blend fibers. The acid
199
treatment was observed to be efficient to remove the surface material resulting in WC that
200
had a clean surface morphology compared to untreated wheat straw having the size of ~30-
201
150 μm as shown in Fig. 2(a). The overview and detailed morphology of the WC are shown
202
in Fig. 2(b) and (c) respectively. It can be seen that the WC appears as aggregates of many
[9]
203
crystalline cellulose fibrils having the diameter of ~ 700-900 nm on the surface. The surface
204
of WC showed the agglomeration of several hundreds of individual cellulose nanofibrils, as
205
similar to a report discussed with microcrystalline cellulose (Mathew, Oksman, & Sain,
206
2005).
207
indicating the smooth morphology of WC in the PCL matrix to form beadless fibers having a
208
diameter range of ~100-400 nm. However, some authors have also reported the appearance
209
of cellulose nanocrystals on the surface of reinforced PVA nanofibers (Peresin, Habibi,
210
Zoppe, Pawlak, & Rojas, 2010). L-Carvone loaded PCL-WC nanofibers are shown in Fig.
211
2(e). However, the smooth and bead-free fibers were obtained and the diameter of the L-
212
Carvone loaded blend fibers were found to be ~ 200-400 nm which was found to be slightly
213
increased compared to the PCL-WC nanofibers.
Fig. 2(d) shows the typical SEM image of electrospun PCL-WC blend fibers
214 215
216 217
Fig. 2. SEM micrographs of (a) Untreated wheat straw fibers (b) Over view of WC at 200
218
μm (c) WC at 20 μm (d) PCL-WC nanofibers (e) Carvone loaded PCL-WC blend fibers.
219 220
3.4
ATR-FOURIER TRANSFORM INFRARED SPECTROSCOPY
[10]
221
ATR-Fourier Transform Infrared Spectroscopy was used to confirm the
222
functional groups and identify the possible interactions between the WC and PCL in the
223
electrospun fibers as shown in Fig. 3. The hydrophilic nature of WC is clearly reflected from
224
the broad absorption band in the range of 3500-3300 cm-1 corresponding to H-bonded OH
225
stretching vibration as shown in Fig. 3(a). The peaks at 2926 and 2864 cm-1 are assigned to
226
aliphatic saturated C-H stretching and aromatic C-H vibrations respectively. The absorption
227
peak at 1734 cm-1 attributed to either acetyl and uronic ester groups of hemicellulose or the
228
ester linkage of lignin or hemicellulose were not observed in the spectrum of WC, in contrast
229
to the research reports (Alemdar, & Sain, 2008; Kaushik, & Singh, 2011; Rao, Jeyapal, &
230
Rajiv, 2014). Hence in the present study, complete removal of lignin and hemicellulose from
231
the extracted WC had occurred due to the acid hydrolysis. The peak at 1634 cm-1 is a
232
characteristic peak of the bending mode of absorbed water. The absorption peaks at 1434
233
and 1371 cm-1 are assigned to CH group deformation (Sun, F., Xu, Sun, R.C., Fowler, &
234
Baird, 2005). The peaks at 1158 and 1033 cm-1 could be assigned to the C-O-C and C-O
235
stretching vibrations. As in Fig. 3(b), the ATR-FTIR spectrum of pure PCL nanofiber shows
236
a strong C=O absorption peak at 1723 cm-1 and medium absorption peaks at 2938 and 2864
237
cm-1 respectively due to the C-H stretching vibration.
238
similar peaks were obtained with slight shifts in the range as shown in Fig. 3(c). The peak at
239
3300 cm-1 of WC was broadened and the intensity of the peak at 1723 cm-1 of PCL was
240
lowered in the PCL-WC nanofibers indicating the presence of strong interaction between OH
241
groups of WC and C=O groups of PCL. Fig. 3(d) shows the L-Carvone loaded PCL-WC
242
fibers. The characteristic absorption peaks of L-Carvone appeared at 2922, 1672, 1434,
243
1371 and 907 cm-1 (Ramamoorthy, & Rajiv, 2014) which confirmed the presence of L-
244
Carvone in the PCL-WC fibers. The much broadened peaks at 1723 and 3327 cm-1 were
245
observed due to the strong adsorption or inter penetration of Carvone within the PCL-WC
246
fibers.
In the case of PCL-WC blend fibers,
[11]
247 248
Fig. 3. ATR-FTIR spectra of (a) Wheat cellulose (WC) (b) Pure PCL
249
nanofibers (d) L-Carvone loaded PCL-WC blend fibers
250
3.5
(c)
PCL-WC
X-RAY DIFFRACTION ANALYSIS
251
The crystalline nature of WC and electrospun nanofibers were analysed using X-ray
252
diffraction analysis as reported in Fig. 4. In Fig. 4a, the broad peaks were observed at 2
253
= 16.7°, 22.5°, and 34.6° corresponding to (101), (002) and (040) planes respectively, which
254
are characteristic of the crystalline structure of cellulose-I type (Chen, Liu, Chang, Cao, &
255
Anderson, 2009; Chena et al., 2011; Park, Baker, Himmel, Parilla, & Johnson, 2010). The
256
characteristic crystalline peaks of PCL nanofibers are reported at 2 = 21.3° and 23.6° for
257
(110) and (200) reflections respectively (Ramamoorthy, & Rajiv, 2014). The effect of
258
incorporation of WC on the crystallinity of PCL nanofibers are shown in Fig. 4b. In the case
259
of PCL-WC blend fibers, the peaks at 2 = 21.5° and 23.7° are the most prominent peaks
260
showing the crystalline nature of PCL. However, the peak at 2 = 22.5° was not visible, but a
261
shoulder like hump was identified at 2 = 22.1° which is an indicative of the presence of
262
cellulose-I type in the PCL-WC fibers as reported in the fibers of poly lactic acid (PLA) and
263
microcrystalline cellulose (Mathew, Oksman, & Sain, 2005).
264
further confirm the interaction between C=O groups of PCL with the -OH groups of WC. The
These diffraction patterns
[12]
265
peaks at 2 = 16.7° and 34.6° were not prominent and were very weak in the blend fibers
266
showing the decrease of crystallinity upon incorporation of WC to PCL nanofibers. The
267
loading of L-Carvone although did not show any considerable effect on the degree of
268
crystallinity of PCL-WC blend fibers as shown in Fig. 4c.
269 270
Fig. 4. XRD patterns of (a) Wheat cellulose (WC) (b) PCL-WC nanofibers (c) L-Carvone
271
loaded PCL-WC blend fibers
272
3.6
THERMOGRAVIMETRIC ANALYSIS
273
The thermal stability of prepared WC, electrospun pure PCL and blend fibers were
274
examined by TGA and DTGA as shown in Fig. 5(i) and (ii) respectively. It can be observed
275
from the Fig. 5i(a), that there was a first weight loss up to 120oC which indicates removal of
276
moisture and solvents. The WC started to decompose at 230oC and the maximum weight
277
loss occurred at 331oC and no additional peaks were observed which confirmed the purity of
278
the isolated cellulose. After heating to 600oC, relatively small amount of solid residue (
279
6.2%) was obtained which may be due to the carbonaceous materials in wheat straw in N2
280
atmosphere (Hornby, Hinrichsen, & Tarverdi, 1997). As seen from Fig. 5ii(b), in pure PCL
281
thermorgram, there was no weight loss up to 350oC and a major weight loss was observed
282
at 416 oC which corresponds to the decomposition of polymer chain. It can be seen from
[13]
283
Fig. 5ii(c) that two stages of thermal degradation occurred for PCL-WC blend fibers. Initial
284
degradation appears at 345oC due to the decomposition of cellulose crosslinked with C=O
285
bonds of PCL. Final degradation occurred at 418oC owing to the complete breakdown of
286
PCL main chain. However, with an addition of L-Carvone to the PCL-WC nanofibers, the
287
thermal degradation was found to decrease slightly to 336oC and 416oC respectively, as in
288
Fig. 5ii(d). It could be observed that the thermal stability of the PCL-WC blend fibers were
289
not very much altered compared to the pure PCL nanofibers while incorporating WC to
290
produce the PCL-WC blend nanofibers. These results are in good agreement with the XRD
291
measurements.
292 293
Fig. 5. (i) TGA thermograms of (a) Wheat cellulose (WC) (b) Pure PCL (c) PCL-WC (d) L-
294
Carvone loaded PCL-WC fibers (ii) DTGA thermograms of (a) Wheat cellulose (WC) (b)
295
Pure PCL (c) PCL-WC (d) L-Carvone loaded PCL-WC fibers
296
3.7.
DIFFERENTIAL SCANNING CALORIMETRY
297
The thermal transitions of WC and blend electrospun fibers were investigated by the
298
DSC measurements as shown in Fig. 6. In Fig. 6a, the DSC thermogram of WC showed an
299
endothermic peak at 68oC, corresponding to water evaporation. After water evaporation, a
300
sharp and clear fusion peak at 326oC appeared due to the fusion of crystalline region of
301
cellulose corresponding to the scission of the glycosidic bonds, with laevoglucose formation.
[14]
302
Fig. 6b and c show the DSC thermograms of electrospun PCL-WC fibers. The thermal
303
properties and crystallinity of the blend fibers are shown in Table 1. Both PCL-WC blend
304
fibers and L-Carvone loaded PCL-WC blend fibers showed only one melting peak indicating
305
that the addition of WC did not affect the arrangement of PCL chains. Similar results were
306
reported with microcrystalline cellulose reinforced PCL composites by injection moulding
307
method (Sabo, Jin, Stark, & Ibach, 2013). The melting temperature and crystallinity (%)
308
were found to be slightly lower than that of the pure PCL nanofibers showing 61 oC and
309
56.1% (Ramamoorthy, & Rajiv, 2014). This could be attributed to the strong interaction of
310
OH group of WC and C=O group of PCL polymer similar to the results reported in the case
311
of PCL-graft-DEM and PCL-Starch blends (Chin-San Wu, 2003; Sugih, Drijfhout, Picchioni,
312
Janssen, & Heeres, 2009).
313
incorporation of L-Carvone could be due to the plasticizing effect of carvone in the PCL-WC
314
nanofibers (De Oliveira Mori, et al., 2015).
315
The further decrease in crystallinity of blend fiber upon
[15]
316
Fig. 6. DSC thermograms of (a) Wheat cellulose (WC) (b) PCL-WC nanofibers (c) L-
317
Carvone loaded PCL-WC blend fibers
318
3.8
SWELLING TEST AND CONTACT ANGLE MEASUREMENTS
319
Swelling tests were carried out to analyse the water absorption of the prepared
320
PCL-WC blend fibers. As shown in Fig. 7(a), the swelling (%) value of pure PCL and the
321
PCL-WC fibers showed 70% and 166% respectively, which confirmed the conversion of
322
hydrophobic PCL to hydrophilic blend fiber. As a result, more of L-Carvone could be sorbed
323
by the PCL-WC fiber which makes the scaffold suitable for the antimicrobial textile
324
applications.
325
To confirm the hydrophilic character of PCL-WC blend, contact angles of water drop
326
on the pure PCL and blend fiber were measured as shown in Fig. 7(b)-(d). It can be seen
327
that the contact angle of pure PCL and PCL-WC blend fibers were found to be 99.2 o and
328
25.6o at 10 s respectively, confirming the hydrophilicity of the blend fibers. Fig. 7(b) shows
329
the contact angle measurements of the pure PCL and PCL-WC blend fibers at various time
330
intervals. The contact angle images of pure PCL and PCL-WC fiber were shown in Fig. 7(c)
331
and (d), respectively. The improvement in the hydrophilic character of PCL-WC blend could
332
be due to the OH groups available on the WC forming a strong interaction with the C=O
333
groups of PCL fibers. These results are in agreement with the results obtained by ATR-FTIR
334
and TGA measurements. Similar results were observed in the research report discussing
335
the hydrophilic nature of PCL-grafted microfibrillated cellulose fibers (Lonnberg, Larsson,
336
Lindstrom, Hult, & Malnstrom, 2011).
337
[16]
338 339
Fig. 7. (a) Swelling test
340
electrospun nanofibers (c) Contact angle image of Pure PCL (d) Contact angle image of
341
PCL-WC fibers at 10 s
(b) Variation of Contact angle measurement with time for
342 343
3.9
344
CARVONE LOADED BLEND FIBER
IN-VITRO RELEASE KINETICS AND ANTIBACTERIAL ACTIVITY OF THE L-
345
The loading efficiency (%) of L-Carvone in the PCL-WC blend fibers were found from
346
an average of triplicate measurements and found to be 70%. The in-vitro release of L-
347
Carvone loaded in the blend fiber was determined as shown in Fig. 8i(a). It could be noticed
348
that burst release of L-Carvone was observed in the first 30 minutes. This could be due to
349
the small amount of loosely bound L-Carvone present on the surface of the blend fibers. The
350
hydrophilic character of PCL-WC fibers are more suitable for the enhanced release rate of L-
351
Carvone. After initial burst release, it was followed by a controlled and sustained release
352
over a period of 30 h. The maximum L-Carvone release (%) from the PCL-WC blend fibers
[17]
353
was found to ~ 84.3 % compared to our previous work reported as 48 % for pure PCL
354
nanofibers (Ramamoorthy, & Rajiv, 2014). The in-vitro release was measured even after 30
355
h in order to confirm the constant release of L-Carvone from the blend fibers. Hence, these
356
release behaviour shows that the prepared electrospun PCL-WC blend fibers could be used
357
as an ideal scaffold for the delivery of hydrophobic essential oil such as L-Carvone.
358
The in-vitro release of L-Carvone from the PCL-WC blend scaffold was
359
studied by the four different kinetic models such as zero order, first order, Higuchi and
360
Korsmeyer Peppas model equations. Fig. 8i (b)-(e) shows the kinetic modelling of the in-vitro
361
release of L-Carvone from the blend fibers. It could be observed that the in-vitro release of L-
362
Carvone followed the Korsmeyer Peppas release kinetic model with a highest value of
363
regression coefficient (R2 = 0.9848) value. The ‘n’ value of the Korsmeyer Peppas model
364
was found to be 0.42, indicating that the release of L-Carvone occurred by the diffusion
365
mechanism.
366
The antibacterial activities of the L-Carvone loaded blend fibers were tested against
367
Staphylococcus aureus and Escherichia coli microorganisms as shown in Fig. 8ii(a) and (b).
368
PCL-WC nanofibers without L-Carvone addition was used as control and the result showed
369
that there was no clear zone inhibition formed for the control samples. The zone of inhibition
370
of L-Carvone loaded PCL-WC fibers was found to be around 22 mm for S.aureus and 21
371
mm for E. coli respectively, which proves the usage of the PCL-WC fibers as an fragrant
372
antimicrobial scaffold.
373 374
[18]
375 376 377 378
Fig. 8(i). (a) In-vitro release of L-Carvone from PCL-WC blend fibers (b)-(e) Kinetic modelling of the in-vitro release of L-Carvone from the PCL-WC fibers
[19]
379 380
Fig. 8(ii). Antibacterial activities of the L-Carvone (5 wt%) loaded PCL-WC (13:3 wt%) blend
381
fibers against (a) Staphylococcus aureus (b) Escherichia coli
382 383
4.0
CONCLUSION
384
Electrospinning is an easier, simple and widely used effective method to produce
385
ultrathin fibers for various applications. In this present work, cellulose from WC was
386
extracted and characterised. The extracted WC was successfully incorporated to obtain
387
PCL-WC blend nanofibers through electrospinning. Further, an effective entrapment of L-
388
Carvone into the PCL-WC blend was carried out. The prepared blend fibers were well
389
characterised by the ATR-FTIR, SEM and XRD techniques. The hydrophilic character of the
390
PCL-WC
391
stability of the PCL-WC fibers was not very much affected after the loading of L-Carvone.
392
Although the release of essential oil are associated with many factors such as volatile
393
nature, activity and degradation of oil, polymer matrix nature, the prepared PCL-WC
394
nanofibers showed ~84.3 % release rate of L-Carvone. Further the Korsmeyer Peppas
395
kinetic modelling confirmed the diffusion mechanism of L-Carvone release from the blend
nanofibers was confirmed by the contact angle measurements. The thermal
[20]
396
fibers. The antibacterial activities of the prepared blend fibers suggested the suitability and
397
applicability of the prepared PCL-WC nanofibers for use as an ideal fragrant formulation of
398
L-Carvone for antimicrobial textile applications.
399
ACKNOWLEDGEMENT
400
Ramamoorthy Manjula acknowledges M.S.A.J. College of Engineering for their
401
support. The instrumentation facility provided under FIST-DST and DRS-UGC to Department
402
of Chemistry, Anna University, Chennai are gratefully acknowledged.
403 404
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24. Moran, J.I, Alvarez, V.A, Cyras, V.P, & Vazquez, A. (2008). Extraction of
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Delivery System Of Liposomally Encapsulated Volatile Oil Of Anethum Graveolens.
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Farmacia, 61, 2.
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27. Park, S., Baker, J.O., Himmel, M.E., Parilla, P.A., & Johnson, D.K. (2010). Cellulose
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crystallinity index: measurement techniques and their impact on interpreting cellulase
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performance. Biotechnology for Biofuels, 3, 10.
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28. Peppas, N.A., & Am Ende, D.J. (1997). Controlled Release of Perfumes from
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Polymers. II. Incorporation and Release of Essential oils from Glossy Polymers.
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Journal of Applied Polymer Science, 66, 509-513.
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29. Peresin, M.S., Habibi, Y., Zoppe, J.O., Pawlak, J.J., & Rojas, O.J. (2010). Nanofiber
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Composites of Polyvinyl alcohol and Cellulose Nanocrystals: Manufacture and
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Characterisation. Biomacromolecules, 11,674-681.
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30. Ramamoorthy, M., & Rajiv, S. (2014). L-carvone-loaded nanofibrous membrane as a
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fragrance delivery system: fabrication, characterization and in vitro study. Flavour
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31. Rao, S.S., Jeyapal, S.G., & Rajiv, S. (2014). Biodegradable electrospun
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cellulose. Composites: Part B, 60, 43-48.
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Polycaprolactone/Microcystalline
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Degradation, Bioresources, 8(3), 3322-3335.
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Gastroretentive Controlled Release Polymeric Suspensions Containing Ciprofloxacin
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essential oils active against indianmeal moth. Boletin de Sanidad Vegetal Plagas,
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30,125-132.
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36. Soliman, E.A., El-Moghazy, A.Y., El-Din, M.S.M., & Massoud, M.A. (2013).
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Microencapsulation of Essential Oils within Alginate: Formulation and in Vitro
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Evaluation of Antifungal Activity. Journal of Encapsulation and Adsorption Sciences,
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3, 48-55.
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37. Sugih, A.K., Drijfhout, J.P., Picchioni, F., Janssen, L.P.B.M., & Heeres, H.J. (2009).
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Synthesis and Properties of Reactive Interfacial Agents for Polycaprolactone-Starch
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Blends. Journal of Applied Polymer Science,114, 2315–2326.
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38. Sun, F., Xu F., Sun, R.C., Fowler, P., & Baird, M.S. (2005). Characteristics of
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Research, 340(1), 97–106.
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cellulose obtained from steam-exploded wheat straw. Carbohydrate
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Fabrication of Electrospun Poly L-lactide and Curcumin Loaded Poly L-lactide
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Nanofibers for Drug Delivery. Fibers and Polymers, 13 (7), 823-830.
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Engineering. Expert Opinion on Biological Therapy, 13 (4), 527-540.
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42. Zhao, L., Wei, Y., Mei, Y., Yang, L., You, Y., Yang, X., & Jiang, Y. (2012).
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Preparation and in Vitro Drug Release Evaluation of Once-Daily Metformin
534
Hydrochloride Sustained-Release Tablets. Pharmacology & Pharmacy, 3, 468-473.
535 536 537
43. Table 1. Thermal properties of electrospun blend nanofibers
538
Name of the sample
Tm (oC)
∆Hf (J/g)
Xc (%)
PCL-WC nanofibers
60.8
42.3
30.32
Carvone loaded PCL-WC nanofibers
60.2
35.4
25.38
S0144-8617(15)00644-X http://dx.doi.org/doi:10.1016/j.carbpol.2015.07.015 CARP 10117
To appear in: Received date: Revised date: Accepted date:
3-3-2015 17-6-2015 2-7-2015
Please cite this article as: Manjula, Ramamoorthy., & Rajiv, Sheeja., IN-VITRO RELEASE OF FRAGRANT L-CARVONE FROM ELECTROSPUN POLY ( − CAPROLACTONE)/WHEATCELLULOSESCAFFOLD.CarbohydratePolymershttp : //dx.doi.org/10.1016/j.carbpol.2015.07.015 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.
[1]
1
IN-VITRO RELEASE OF FRAGRANT L-CARVONE FROM ELECTROSPUN POLY (ɛ-
2
CAPROLACTONE) / WHEAT CELLULOSE SCAFFOLD
3
Ramamoorthy Manjula and Sheeja Rajiv*
4
Department of Chemistry, Anna University, Chennai, Tamilnadu 600 025, India.
5
*Corresponding author: Dr.Sheeja Rajiv
6
E-mail: [email protected] ; Tel: +914422358658, Fax: +914422200889
7 8 9
HIGHLIGHTS
10 11
L-Carvone loaded PCL /Wheat cellulose (WC) nanofibers were prepared by the electrospinning technique which showed good antimicrobial activity.
The in-vitro release of L-Carvone from the PCL-WC fibers was found to follow
12
Korsmeyer Peppas kinetic model which indicated diffusion controlled mechanism of
13
release.
14 15 16
Bioactive PCL-WC scaffold is an ideal fragrant scaffold for antimicrobial textile applications.
ABSTRACT
17
The release kinetics of L-Carvone loaded from electrospun Poly (ɛ-caprolactone)
18
(PCL) and Wheat cellulose (WC) blend were studied. WC was extracted from wheat straw, a
19
cost effective agricultural waste by the acid hydrolysis method. A homogeneous solution of
20
PCL-WC (13:3 wt%) was optimised to produce beadless electrospun PCL-WC blend
21
nanofibers. Further, WC and the prepared electrospun PCL-WC blend fibers were
22
systematically characterised by ATR-FTIR,
23
measurements. The hydrophilic character of the blend fibers was analysed using swelling
24
tests and contact angle measurements. The loading efficiency of L-Carvone into the
25
electrospun PCL-WC blend fibers was evaluated to be ~ 70%. The in-vitro release of L-
26
Carvone from PCL-WC blend fibers followed Korsmeyer Peppas kinetic model indicating
SEM, XRD, TGA, DTGA, and DSC
[2]
27
the diffusion mechanism and the maximum release of L-Carvone was found to be ~ 84%
28
over a period of 30 h. These results would offer the prepared PCL-WC blend as an ideal
29
fibrous mesh for fragrant antimicrobial textile applications.
30 31
KEY WORDS: L- Carvone; Wheat Cellulose; Blend; Poly (ɛ-caprolactone); Release kinetics;
32
Korsmeyer Peppas model
33 34 35
1.0
INTRODUCTION
36
Over the past few decades, electrospun biocomposite nanofibers are of great
37
importance, owing to their unique properties like high porosity, large surface area, small pore
38
size, superior mechanical and thermal properties (Miao et al., 2012).
39
cellulose-based nanofibers, nanoparticles, nanocrystals are reported to provide many
40
advantages comprising world wide availability from various non-conventional resources, low
41
cost, better biodegradability and easy tailor made process ability (Azizi Samir, Alloin, &
42
Dufresne, 2005; Hou, Zhou, & Wang, 2009). The use of natural cellulose fibers consist of a
43
major research work in the production of biodegradable composites. The reinforcement of
44
cellulose nanoparticles has attracted significant attention due to their abundance, renewable
45
nature, large surface area and good mechanical strength (Chazeau, Cavaille, Canova,
46
Dendievel, & Boutherin, 1999; Choi, & Simonsen, 2006; Helbert, Cavaille, & Dufresne,
47
1996; Wang, Sain, & Oksman, 2007).
Cellulose and
48
Essential oils have excellent antibacterial, antifungal, antioxidant, insect-repellent and
49
insecticidal properties (Bilia, Guccione, Isacchi, Righeschi, Firenzuoli, & Bergonzi, 2014;
50
Fukumoto, Sawasaki, Okuyama, Miyake, & Yokogoshi, 2006). However, the highly volatile
51
nature and degradation upon exposure to sunlight limits their successful application. Recent
[3]
52
researchers made several formulations in order to achieve chemical stability, a controlled
53
release, increased efficiency and activity of essential oil. To ensure safer and easier
54
handling of essential oils, they are entrapped into solid carriers which retain the bioactive
55
compounds and further enables controlled release of the ingredients thereby enhancing their
56
bioavailability and efficacy (Ortan, Ferdes, Rodino, Pirvu, &
57
biodegradable and biocompatible systems have been reported for the encapsulation and
58
sustained release of essential oils (Dimaa, Gitina, Alexea, & Dimab, 2013; Ghosh,
59
Mukherjee, & Chandrasekaran, 2013; Ortan, Campeanu, Dinu–Pirvu, & Popescu, 2009;
60
Sanna passion, Bazzoni, & Moretti, 2004; Shirwaikar, Prabhu, & Kumar, 2008; Soliman, El-
61
Moghazy, El-Din, & Massoud,. 2013). PCL is one of the excellent biodegradable and semi
62
crystalline linear hydrophobic polymers, which find many applications in biomedical field due
63
to their good biocompatibility, superior mechanical properties and complete degradation to
64
non-toxic by-products (Zhang, Gupte, & Ma, 2013). Moreover, PCL nanofiber matrices
65
have slower degradation rates among the well-known biodegradable synthetic polyesters
66
such as PGA, PLGA and PLA due to the presence of five hydrophobic –CH2 moieties in the
67
repeating units (Hao, Yuan, & Deng, 2002; Kanani, & Bahrami, 2011). The main aim of the
68
present study was to fabricate the L-Carvone loaded PCL-WC electrospun blend fibers using
69
electrospinning method. The cellulose extracted from wheat straw fibers were added to PCL
70
to improve the biodegradability and wettability of PCL thereby making the scaffold suitable
71
as an antimicrobial fragrant scaffold. A homogeneous solution of PCL-WC was prepared and
72
electrospun to obtain PCL-WC blend fibers. Further, the L-Carvone was blended with the
73
PCL-WC solution and electrospun to form the L-Carvone loaded PCL-WC blend fibers. The
74
kinetics of the ‘in-vitro’ release of L-Carvone from the blend fibers was fitted with four
75
different kinetic models. In addition, the essential oil was highly active even after the release
76
from the electrospun fibers which were confirmed by their antimicrobial activity against gram-
77
positive and gram-negative microorganisms.
78 79
2.0
MATERIALS AND EXPERIMENTAL METHODS
Draganescu, 2013). Many
[4]
80
2.1
MATERIALS
81
Wheat straw used in the present work was received from Dharmapuri, Tamil Nadu,
82
India. PCL (Mn ~ 70000 – 90000) and L-Carvone ( 97%) were obtained from Sigma-Aldrich,
83
India. Chloroform, Trifluoroacetic acid (TFA), Ethanol, Sodium hydroxide (NaOH), Sulphuric
84
acid (H2SO4), Hydrogen peroxide (H2O2), Nitric acid (HNO3) were purchased from Fischer
85
Scientific Company. All the reagents used were of analytical grade.
86 87 88 89
2.2
EXTRACTION OF CELLULOSE FROM WHEAT STRAW
90
Initially, wheat straw was washed with distilled water to remove impurities
91
covering the external cell wall. The baled wheat straw was cut manually to 1-2 cm. The
92
cut wheat straw was pre-treated
93
agitation to remove lignin (Alemdar & Sain 2008). The extraction of wheat cellulose (WC)
94
was carried out according to the procedure reported for extraction of cellulose from sisal
95
fibers (Moran, Alvarez, Cyras, & Vazquez, 2008).
with 3% NaOH at 50oC for 2 h under continuous
96 97
2.3
FABRICATION OF ELECTROSPUN PCL-WC BLEND FIBRES
98
The homogeneous solutions of 13 wt% PCL and 3 wt% WC were prepared using
99
chloroform and TFA solvents respectively. Then, PCL-WC blend solution was prepared by
100
mixing the solution of WC to the PCL solution over a period of 30 min until the solution
101
became homogeneous. The polymer blend solution was taken in a 5 ml syringe having a
102
needle tip of 0.6 mm inner diameter. A high voltage of 25 kV was supplied directly from a
103
high DC voltage power supply to the needle and the negative terminal of the power was
104
connected to the collector covering with an aluminium foil. The polymer solutions were
105
electrospun at a flow rate of 0.9 ml/h and the tip-to-collector distance of 20 cm.
106
[5]
107
2.4
PREPARATION OF L-CARVONE LOADED PCL-WC BLEND FIBERS
108
A known weight of sample of standard size (1 x 1 cm) of PCL-WC blend nanofibrous
109
membrane was immersed in a 5 wt% of L-Carvone and allowed to absorb the active agent
110
over a period of 24 h at room temperature of 28oC (Peppas, & Am Ende, 1997).
111 112
2.5 MEASUREMENTS AND CHARACTERISATIONS
113
The size distribution of the extracted wheat cellulose was studied using a Malvern Zeta Sizer
114
Nano-S Version 7.03. The analysis was carried out using water as dispersant at 25oC in
115
order to measure the particle size. Approximately, 250 measurements were taken to
116
measure
117
Spectrophotometer equipped with a diamond crystal at an angle of incidence of 180°) was
118
used to identify the functional groups present in the WC and electrospun PCL-WC blend
119
fibers in the range of 400 to 4000 cm-1. The morphological features of wheat straw, extracted
120
WC and the prepared electrospun nanofibers were studied using Scanning Electron
121
Microscopy (FEI Quanta FEG 200 HRSEM). The samples were sputter coated with gold
122
under a fine coater for 120 s. An accelerating voltage of 10 kV was applied to observe SEM
123
images and the diameters of the isolated nanofibrils were measured using the Adobe
124
Photoshop CS3 Extended Software PS version 10.0 model. X-ray diffraction patterns of the
125
untreated straw, obtained WC and all prepared nanofibers was performed using a X’pert Pro
126
PANalytical Instrument using Cu Kᾳ radiation (λ=1.5418 Ao) in the 2 scale from 5-60o. The
127
thermal degradation characteristics of all samples were examined using thermogravimetric
128
analysis (TGA/DTA Model SDT 2600) at a heating rate of 10oC/min from 35 to 800oC with
129
continuous nitrogen flow of 20 cm3/min. The DSC measurements were performed with all
130
samples under nitrogen atmosphere with a scanning speed of 10oC/min and a heating rate
131
of 10oC/min from 0 to 750oC using DSC Q200 V24.4 Build 116 Model. In addition, the
132
degree of crystallinity (Xc) of PCL-WC nanofibers was calculated by dividing the measured
133
enthalpy of fusion (Hf) from DSC thermogram with the standard enthalpy of fusion for the
the
size
distribution of
the
sample.
ATR-FTIR analysis (Perkin-Elmer
[6]
134
100% crystalline PCL polymer (Hf0 = 139.5 Jg-1) (Crescenzi, Mancini, Calzolari, & Borri,
135
1972). The swelling characteristics of the fibers in deionised water were studied using the
136
reported procedure (Elayaraja, et al., 2011). In addition, the hydrophilicity of the prepared
137
fibers were studied by the contact angle measurements using an Euromex Optical
138
Microscope equipped with a CCD camera (Thangaraju, Srinivasan, Kumar, Sehgal, & Rajiv,
139
2012).
140 141 142 143 144
2.6
DETERMINATION OF LOADING EFFICIENCY OF L-CARVONE
145
The actual quantity of L-Carvone in the PCL-WC fibers was determined using UV-
146
Visible spectrophotometer at 236 nm. The amount of L-Carvone loaded in the blend fibres
147
was determined according to equation (1). All the experiments were performed in triplicates.
148
149
(%) =
× 100
(1)
150 151
2.7
IN-VITRO RELEASE STUDIES OF L-CARVONE AND KINETIC MODELLING
152
A known weight of L-Carvone loaded PCL-WC fibrous mat was placed in a 20 ml vial
153
containing absolute ethanol at 28oC. At regular time intervals, 5 ml of the aliquot was taken
154
and replaced with the same quantity of ethanol. The amount of L-Carvone in the releasing
155
medium was determined using UV-Visible spectrophotometer at the absorption wavelength
156
of 236 nm. The in-vitro release of L-Carvone was fitted into zero order, first order, Higuchi
157
and Korsmeyer peppas equations respectively to study the kinetic mechanism of L-Carvone
158
release from the fibers ( Sahoo, Chakraborti, & Behera, 2012; Zhao et al. 2012).
159
The four different kinetic model equations are as follows:
[7]
Q = Q -
160
Zero order equation :
161
First order equation:
162
Higuchi equation:
163
Korsmeyer – Peppas equation:
164
Where,
165
time t,
166
2.8
,
t
ln = ln Q Q =
(2) t
(3)
/
(4) /
=
(5)
are the release rate constants,
/
is fraction of oil released at
is constant and n is the diffusion constant that represents the release mechanism. ANTIBACTERIAL ACTIVITY OF THE L-CARVONE LOADED PCL-WC FIBERS
167
The antibacterial activities of the PCL-WC scaffolds were studied using gram positive
168
and gram negative, Staphylococcus aureus and Escherichia coli respectively as test
169
pathogens by Kirby- Bauer disk diffusion method. These bacterial pathogens were spread on
170
the L-Carvone loaded fibers in nutrient agar test plates under sterile conditions and
171
incubated for a period of 24 h and the zone of inhibition was measured.
172
3.0
RESULTS AND DISCUSSION
173
3.1
CELLULOSE YIELD
174
The wheat straw raw material was subjected to a delignification process in order to
175
facilitate the removal of lignin (Alemdar & Sain 2008). Initially, the crushed wheat straw fibers
176
were treated with alkali to remove the wax and partial separation of the cellulose fibers from
177
the cell wall. The second treatment with sulphuric acid led to more removal of non-
178
cellulosic substances and
179
treatment with nitric acid increased the crystallinity and molecular weight of cellulose
180
fibers resulting in a good yield and better quality of WC product in the nano scale as
181
reported (Moran et al., 2008).
182
3.2
formed
highly
stable
cellulose fibers. Moreover, further
PARTICLE SIZE DISTRIBUTION OF THE EXTRACTED WC
183
The acid treatment provided a viable and simple method for synthesising
184
nanocellulose. The average particle size of the extracted WC after the chemical treatment
[8]
185
and their average size distributions are shown in Fig. 1. The acid treatment of wheat straw
186
yielded fibrils with a Z-average diameter of 1.0 μm. The average particle size of the
187
extracted cellulose fibrils were found in the diameter range of 712-825 nm. As can be seen
188
from the Fig. 1, WCs were found to be in the diameter range of 825-955 nm with 33.6% of
189
the total intensity. Almost 53.7% of the particles were found to be in the diameters of
190
approximately 712-825 nm and only 12.7% of particles were found to have diameters in the
191
range of 600-712 nm.
192 193
Fig. 1. Particle Size distribution of extracted WC
194 195
3.3
MORPHOLOGY ANALYSIS
196
The SEM images of untreated wheat straw, WC and electrospun blend fibers are
197
shown in Fig. 2(a) – (e). SEM analysis is one of the very familiar method to examine the
198
surface morphology of the extracted cellulose and the electrospun blend fibers. The acid
199
treatment was observed to be efficient to remove the surface material resulting in WC that
200
had a clean surface morphology compared to untreated wheat straw having the size of ~30-
201
150 μm as shown in Fig. 2(a). The overview and detailed morphology of the WC are shown
202
in Fig. 2(b) and (c) respectively. It can be seen that the WC appears as aggregates of many
[9]
203
crystalline cellulose fibrils having the diameter of ~ 700-900 nm on the surface. The surface
204
of WC showed the agglomeration of several hundreds of individual cellulose nanofibrils, as
205
similar to a report discussed with microcrystalline cellulose (Mathew, Oksman, & Sain,
206
2005).
207
indicating the smooth morphology of WC in the PCL matrix to form beadless fibers having a
208
diameter range of ~100-400 nm. However, some authors have also reported the appearance
209
of cellulose nanocrystals on the surface of reinforced PVA nanofibers (Peresin, Habibi,
210
Zoppe, Pawlak, & Rojas, 2010). L-Carvone loaded PCL-WC nanofibers are shown in Fig.
211
2(e). However, the smooth and bead-free fibers were obtained and the diameter of the L-
212
Carvone loaded blend fibers were found to be ~ 200-400 nm which was found to be slightly
213
increased compared to the PCL-WC nanofibers.
Fig. 2(d) shows the typical SEM image of electrospun PCL-WC blend fibers
214 215
216 217
Fig. 2. SEM micrographs of (a) Untreated wheat straw fibers (b) Over view of WC at 200
218
μm (c) WC at 20 μm (d) PCL-WC nanofibers (e) Carvone loaded PCL-WC blend fibers.
219 220
3.4
ATR-FOURIER TRANSFORM INFRARED SPECTROSCOPY
[10]
221
ATR-Fourier Transform Infrared Spectroscopy was used to confirm the
222
functional groups and identify the possible interactions between the WC and PCL in the
223
electrospun fibers as shown in Fig. 3. The hydrophilic nature of WC is clearly reflected from
224
the broad absorption band in the range of 3500-3300 cm-1 corresponding to H-bonded OH
225
stretching vibration as shown in Fig. 3(a). The peaks at 2926 and 2864 cm-1 are assigned to
226
aliphatic saturated C-H stretching and aromatic C-H vibrations respectively. The absorption
227
peak at 1734 cm-1 attributed to either acetyl and uronic ester groups of hemicellulose or the
228
ester linkage of lignin or hemicellulose were not observed in the spectrum of WC, in contrast
229
to the research reports (Alemdar, & Sain, 2008; Kaushik, & Singh, 2011; Rao, Jeyapal, &
230
Rajiv, 2014). Hence in the present study, complete removal of lignin and hemicellulose from
231
the extracted WC had occurred due to the acid hydrolysis. The peak at 1634 cm-1 is a
232
characteristic peak of the bending mode of absorbed water. The absorption peaks at 1434
233
and 1371 cm-1 are assigned to CH group deformation (Sun, F., Xu, Sun, R.C., Fowler, &
234
Baird, 2005). The peaks at 1158 and 1033 cm-1 could be assigned to the C-O-C and C-O
235
stretching vibrations. As in Fig. 3(b), the ATR-FTIR spectrum of pure PCL nanofiber shows
236
a strong C=O absorption peak at 1723 cm-1 and medium absorption peaks at 2938 and 2864
237
cm-1 respectively due to the C-H stretching vibration.
238
similar peaks were obtained with slight shifts in the range as shown in Fig. 3(c). The peak at
239
3300 cm-1 of WC was broadened and the intensity of the peak at 1723 cm-1 of PCL was
240
lowered in the PCL-WC nanofibers indicating the presence of strong interaction between OH
241
groups of WC and C=O groups of PCL. Fig. 3(d) shows the L-Carvone loaded PCL-WC
242
fibers. The characteristic absorption peaks of L-Carvone appeared at 2922, 1672, 1434,
243
1371 and 907 cm-1 (Ramamoorthy, & Rajiv, 2014) which confirmed the presence of L-
244
Carvone in the PCL-WC fibers. The much broadened peaks at 1723 and 3327 cm-1 were
245
observed due to the strong adsorption or inter penetration of Carvone within the PCL-WC
246
fibers.
In the case of PCL-WC blend fibers,
[11]
247 248
Fig. 3. ATR-FTIR spectra of (a) Wheat cellulose (WC) (b) Pure PCL
249
nanofibers (d) L-Carvone loaded PCL-WC blend fibers
250
3.5
(c)
PCL-WC
X-RAY DIFFRACTION ANALYSIS
251
The crystalline nature of WC and electrospun nanofibers were analysed using X-ray
252
diffraction analysis as reported in Fig. 4. In Fig. 4a, the broad peaks were observed at 2
253
= 16.7°, 22.5°, and 34.6° corresponding to (101), (002) and (040) planes respectively, which
254
are characteristic of the crystalline structure of cellulose-I type (Chen, Liu, Chang, Cao, &
255
Anderson, 2009; Chena et al., 2011; Park, Baker, Himmel, Parilla, & Johnson, 2010). The
256
characteristic crystalline peaks of PCL nanofibers are reported at 2 = 21.3° and 23.6° for
257
(110) and (200) reflections respectively (Ramamoorthy, & Rajiv, 2014). The effect of
258
incorporation of WC on the crystallinity of PCL nanofibers are shown in Fig. 4b. In the case
259
of PCL-WC blend fibers, the peaks at 2 = 21.5° and 23.7° are the most prominent peaks
260
showing the crystalline nature of PCL. However, the peak at 2 = 22.5° was not visible, but a
261
shoulder like hump was identified at 2 = 22.1° which is an indicative of the presence of
262
cellulose-I type in the PCL-WC fibers as reported in the fibers of poly lactic acid (PLA) and
263
microcrystalline cellulose (Mathew, Oksman, & Sain, 2005).
264
further confirm the interaction between C=O groups of PCL with the -OH groups of WC. The
These diffraction patterns
[12]
265
peaks at 2 = 16.7° and 34.6° were not prominent and were very weak in the blend fibers
266
showing the decrease of crystallinity upon incorporation of WC to PCL nanofibers. The
267
loading of L-Carvone although did not show any considerable effect on the degree of
268
crystallinity of PCL-WC blend fibers as shown in Fig. 4c.
269 270
Fig. 4. XRD patterns of (a) Wheat cellulose (WC) (b) PCL-WC nanofibers (c) L-Carvone
271
loaded PCL-WC blend fibers
272
3.6
THERMOGRAVIMETRIC ANALYSIS
273
The thermal stability of prepared WC, electrospun pure PCL and blend fibers were
274
examined by TGA and DTGA as shown in Fig. 5(i) and (ii) respectively. It can be observed
275
from the Fig. 5i(a), that there was a first weight loss up to 120oC which indicates removal of
276
moisture and solvents. The WC started to decompose at 230oC and the maximum weight
277
loss occurred at 331oC and no additional peaks were observed which confirmed the purity of
278
the isolated cellulose. After heating to 600oC, relatively small amount of solid residue (
279
6.2%) was obtained which may be due to the carbonaceous materials in wheat straw in N2
280
atmosphere (Hornby, Hinrichsen, & Tarverdi, 1997). As seen from Fig. 5ii(b), in pure PCL
281
thermorgram, there was no weight loss up to 350oC and a major weight loss was observed
282
at 416 oC which corresponds to the decomposition of polymer chain. It can be seen from
[13]
283
Fig. 5ii(c) that two stages of thermal degradation occurred for PCL-WC blend fibers. Initial
284
degradation appears at 345oC due to the decomposition of cellulose crosslinked with C=O
285
bonds of PCL. Final degradation occurred at 418oC owing to the complete breakdown of
286
PCL main chain. However, with an addition of L-Carvone to the PCL-WC nanofibers, the
287
thermal degradation was found to decrease slightly to 336oC and 416oC respectively, as in
288
Fig. 5ii(d). It could be observed that the thermal stability of the PCL-WC blend fibers were
289
not very much altered compared to the pure PCL nanofibers while incorporating WC to
290
produce the PCL-WC blend nanofibers. These results are in good agreement with the XRD
291
measurements.
292 293
Fig. 5. (i) TGA thermograms of (a) Wheat cellulose (WC) (b) Pure PCL (c) PCL-WC (d) L-
294
Carvone loaded PCL-WC fibers (ii) DTGA thermograms of (a) Wheat cellulose (WC) (b)
295
Pure PCL (c) PCL-WC (d) L-Carvone loaded PCL-WC fibers
296
3.7.
DIFFERENTIAL SCANNING CALORIMETRY
297
The thermal transitions of WC and blend electrospun fibers were investigated by the
298
DSC measurements as shown in Fig. 6. In Fig. 6a, the DSC thermogram of WC showed an
299
endothermic peak at 68oC, corresponding to water evaporation. After water evaporation, a
300
sharp and clear fusion peak at 326oC appeared due to the fusion of crystalline region of
301
cellulose corresponding to the scission of the glycosidic bonds, with laevoglucose formation.
[14]
302
Fig. 6b and c show the DSC thermograms of electrospun PCL-WC fibers. The thermal
303
properties and crystallinity of the blend fibers are shown in Table 1. Both PCL-WC blend
304
fibers and L-Carvone loaded PCL-WC blend fibers showed only one melting peak indicating
305
that the addition of WC did not affect the arrangement of PCL chains. Similar results were
306
reported with microcrystalline cellulose reinforced PCL composites by injection moulding
307
method (Sabo, Jin, Stark, & Ibach, 2013). The melting temperature and crystallinity (%)
308
were found to be slightly lower than that of the pure PCL nanofibers showing 61 oC and
309
56.1% (Ramamoorthy, & Rajiv, 2014). This could be attributed to the strong interaction of
310
OH group of WC and C=O group of PCL polymer similar to the results reported in the case
311
of PCL-graft-DEM and PCL-Starch blends (Chin-San Wu, 2003; Sugih, Drijfhout, Picchioni,
312
Janssen, & Heeres, 2009).
313
incorporation of L-Carvone could be due to the plasticizing effect of carvone in the PCL-WC
314
nanofibers (De Oliveira Mori, et al., 2015).
315
The further decrease in crystallinity of blend fiber upon
[15]
316
Fig. 6. DSC thermograms of (a) Wheat cellulose (WC) (b) PCL-WC nanofibers (c) L-
317
Carvone loaded PCL-WC blend fibers
318
3.8
SWELLING TEST AND CONTACT ANGLE MEASUREMENTS
319
Swelling tests were carried out to analyse the water absorption of the prepared
320
PCL-WC blend fibers. As shown in Fig. 7(a), the swelling (%) value of pure PCL and the
321
PCL-WC fibers showed 70% and 166% respectively, which confirmed the conversion of
322
hydrophobic PCL to hydrophilic blend fiber. As a result, more of L-Carvone could be sorbed
323
by the PCL-WC fiber which makes the scaffold suitable for the antimicrobial textile
324
applications.
325
To confirm the hydrophilic character of PCL-WC blend, contact angles of water drop
326
on the pure PCL and blend fiber were measured as shown in Fig. 7(b)-(d). It can be seen
327
that the contact angle of pure PCL and PCL-WC blend fibers were found to be 99.2 o and
328
25.6o at 10 s respectively, confirming the hydrophilicity of the blend fibers. Fig. 7(b) shows
329
the contact angle measurements of the pure PCL and PCL-WC blend fibers at various time
330
intervals. The contact angle images of pure PCL and PCL-WC fiber were shown in Fig. 7(c)
331
and (d), respectively. The improvement in the hydrophilic character of PCL-WC blend could
332
be due to the OH groups available on the WC forming a strong interaction with the C=O
333
groups of PCL fibers. These results are in agreement with the results obtained by ATR-FTIR
334
and TGA measurements. Similar results were observed in the research report discussing
335
the hydrophilic nature of PCL-grafted microfibrillated cellulose fibers (Lonnberg, Larsson,
336
Lindstrom, Hult, & Malnstrom, 2011).
337
[16]
338 339
Fig. 7. (a) Swelling test
340
electrospun nanofibers (c) Contact angle image of Pure PCL (d) Contact angle image of
341
PCL-WC fibers at 10 s
(b) Variation of Contact angle measurement with time for
342 343
3.9
344
CARVONE LOADED BLEND FIBER
IN-VITRO RELEASE KINETICS AND ANTIBACTERIAL ACTIVITY OF THE L-
345
The loading efficiency (%) of L-Carvone in the PCL-WC blend fibers were found from
346
an average of triplicate measurements and found to be 70%. The in-vitro release of L-
347
Carvone loaded in the blend fiber was determined as shown in Fig. 8i(a). It could be noticed
348
that burst release of L-Carvone was observed in the first 30 minutes. This could be due to
349
the small amount of loosely bound L-Carvone present on the surface of the blend fibers. The
350
hydrophilic character of PCL-WC fibers are more suitable for the enhanced release rate of L-
351
Carvone. After initial burst release, it was followed by a controlled and sustained release
352
over a period of 30 h. The maximum L-Carvone release (%) from the PCL-WC blend fibers
[17]
353
was found to ~ 84.3 % compared to our previous work reported as 48 % for pure PCL
354
nanofibers (Ramamoorthy, & Rajiv, 2014). The in-vitro release was measured even after 30
355
h in order to confirm the constant release of L-Carvone from the blend fibers. Hence, these
356
release behaviour shows that the prepared electrospun PCL-WC blend fibers could be used
357
as an ideal scaffold for the delivery of hydrophobic essential oil such as L-Carvone.
358
The in-vitro release of L-Carvone from the PCL-WC blend scaffold was
359
studied by the four different kinetic models such as zero order, first order, Higuchi and
360
Korsmeyer Peppas model equations. Fig. 8i (b)-(e) shows the kinetic modelling of the in-vitro
361
release of L-Carvone from the blend fibers. It could be observed that the in-vitro release of L-
362
Carvone followed the Korsmeyer Peppas release kinetic model with a highest value of
363
regression coefficient (R2 = 0.9848) value. The ‘n’ value of the Korsmeyer Peppas model
364
was found to be 0.42, indicating that the release of L-Carvone occurred by the diffusion
365
mechanism.
366
The antibacterial activities of the L-Carvone loaded blend fibers were tested against
367
Staphylococcus aureus and Escherichia coli microorganisms as shown in Fig. 8ii(a) and (b).
368
PCL-WC nanofibers without L-Carvone addition was used as control and the result showed
369
that there was no clear zone inhibition formed for the control samples. The zone of inhibition
370
of L-Carvone loaded PCL-WC fibers was found to be around 22 mm for S.aureus and 21
371
mm for E. coli respectively, which proves the usage of the PCL-WC fibers as an fragrant
372
antimicrobial scaffold.
373 374
[18]
375 376 377 378
Fig. 8(i). (a) In-vitro release of L-Carvone from PCL-WC blend fibers (b)-(e) Kinetic modelling of the in-vitro release of L-Carvone from the PCL-WC fibers
[19]
379 380
Fig. 8(ii). Antibacterial activities of the L-Carvone (5 wt%) loaded PCL-WC (13:3 wt%) blend
381
fibers against (a) Staphylococcus aureus (b) Escherichia coli
382 383
4.0
CONCLUSION
384
Electrospinning is an easier, simple and widely used effective method to produce
385
ultrathin fibers for various applications. In this present work, cellulose from WC was
386
extracted and characterised. The extracted WC was successfully incorporated to obtain
387
PCL-WC blend nanofibers through electrospinning. Further, an effective entrapment of L-
388
Carvone into the PCL-WC blend was carried out. The prepared blend fibers were well
389
characterised by the ATR-FTIR, SEM and XRD techniques. The hydrophilic character of the
390
PCL-WC
391
stability of the PCL-WC fibers was not very much affected after the loading of L-Carvone.
392
Although the release of essential oil are associated with many factors such as volatile
393
nature, activity and degradation of oil, polymer matrix nature, the prepared PCL-WC
394
nanofibers showed ~84.3 % release rate of L-Carvone. Further the Korsmeyer Peppas
395
kinetic modelling confirmed the diffusion mechanism of L-Carvone release from the blend
nanofibers was confirmed by the contact angle measurements. The thermal
[20]
396
fibers. The antibacterial activities of the prepared blend fibers suggested the suitability and
397
applicability of the prepared PCL-WC nanofibers for use as an ideal fragrant formulation of
398
L-Carvone for antimicrobial textile applications.
399
ACKNOWLEDGEMENT
400
Ramamoorthy Manjula acknowledges M.S.A.J. College of Engineering for their
401
support. The instrumentation facility provided under FIST-DST and DRS-UGC to Department
402
of Chemistry, Anna University, Chennai are gratefully acknowledged.
403 404
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405
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43. Table 1. Thermal properties of electrospun blend nanofibers
538
Name of the sample
Tm (oC)
∆Hf (J/g)
Xc (%)
PCL-WC nanofibers
60.8
42.3
30.32
Carvone loaded PCL-WC nanofibers
60.2
35.4
25.38