- Email: [email protected]
Development of grafted xyloglucan micelles for pulmonary delivery of curcumin: In vitro and in vivo studies
Accepted Manuscript Title: Development of grafted xyloglucan micelles for pulmonary delivery of curcumin: In vitro and in vivo studies Author: Hitendra S. Mahajan Prashant R. Mahajan PII: DOI: Reference:
S0141-8130(15)00668-6 http://dx.doi.org/doi:10.1016/j.ijbiomac.2015.09.053 BIOMAC 5389
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
23-7-2015 28-8-2015 27-9-2015
Please cite this article as: H.S. Mahajan, P.R. Mahajan, Development of grafted xyloglucan micelles for pulmonary delivery of curcumin: In vitro and in vivo studies, International Journal of Biological Macromolecules (2015), http://dx.doi.org/10.1016/j.ijbiomac.2015.09.053 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.
Development of grafted xyloglucan micelles for pulmonary delivery of
2
curcumin: In vitro and in vivo studies
3
Hitendra S Mahajan*, Prashant R Mahajan
4
R.C. Patel Institute of Pharmaceutical Education and Research Shirpur, Dist Dhule.
cr
ip t
1
us
5 6
an
7
M
8
d
9
*corresponding author
11
Dr. Hitendra S Mahajan
12
R. C. Patel Institute of Pharmaceutical Education & Research,
13
Near Karvand Naka, Shirpur-425405,
14
Dist: Dhule, Maharashtra, India.
15
E mail- [email protected]
16
M.No – 91-9423487043,
17
Phone- 91-2563255189 (office)
18
Fax – 91-2563255180
Ac ce p
te
10
19 1
Page 1 of 30
Abstract
21
A novel grafted copolymer consisting of L-lactide grafted xyloglucan was synthesized by
22
polymerization reaction and characterized. The grafted copolymers were analyzed by
23
Fourier-transform infrared spectrometry (FT-IR) and 1H nuclear magnetic resonance (1H
24
NMR) was performed to confirm the grafting of L-poly lactic acid on xyloglucan. The
25
grafted polymer forms micelles at the critical micelle concentration of 0.0150 wt% with the
26
average particle size of 102 nm, as determined by particle size analyzer. The zeta
27
potential of the curcumin loaded micelles was -18.2 mV, an acceptable drug loading
28
efficiency of 68.9 ± 0.02% and the entrapment efficiency of 96.38± 0.2%. The release
29
study for 5h showed a sustained release property. In vitro assessment demonstrates
30
suitability of micelles as dry powder for inhalation. In vivo studies showed significant
31
improvement in bioavailability on pulmonary administration of curcumin micelles as DPI
32
formulation. The potential for pulmonary delivery curcumin loaded in micelles was
33
evaluated. In conclusion, polymeric micelle based on a newly synthesized grafted
34
xyloglucan could be suitable carrier for pulmonary delivery of curcumin.
35
Key words- Grafted polymer, xyloglucan, micelles, drug release, curcumin
36
1. Introduction
37
Naturally occurring carbohydrate polymers are obtained from the plant, animal and
38
microbial sources. Recently, much consideration has been given to chemical modification
39
of natural polymers (Kumar, et al 2009, Mishra, et al 2010; Tripathy, et al 2009).
40
Amphiphilic copolymers, composed of hydrophilic and hydrophobic moieties, can form
41
micelles spontaneously or other self-aggregates in an isotropic aqueous solution. These
Ac ce p
te
d
M
an
us
cr
ip t
20
2
Page 2 of 30
polymeric self-aggregates have been recognized as a potential delivery carrier as the
43
water insoluble core can serve as a reservoir for different hydrophobic agents. These
44
amphiphilic copolymers usually consist of biocompatible, biodegradable hydrophobic
45
polymer blocks such as polyesters or poly (amino acids) covalently bonded to a
46
biocompatible hydrophilic block such as polyethylene glycol or natural polysaccharides.
47
(Letchford and Burt, 2007).
48
Seed xyloglucan (XG) is a natural, water-soluble and non toxic polysaccharide. It has
49
cross-linked conformation and consists of a (1→4)-β-D-glucan backbone and (1→6)-α-D-
50
xylose branches that are partially substituted by (1→2)-β-D-galactoxylose (Carpita and
51
Gibeaut, 1993). Xyloglucan play important roles in the control of cell expansion in the
52
seeds of many dicotyledons (Buckeridge, et al 2003). These seed xyloglucans obtained
53
from seeds of Tamarindus indica had a numerous applications as common additives for
54
food and cosmetic, where they act as thickeners and stabilizing agents (Rao and
55
Srivastava, 1973, Maeda, et al, 2007). Xyloglucan has been studied as a potential drug
56
delivery carrier because it is non-toxic, non-mutagenic, non-irritant, blood compatible and
57
biodegradable (Avachat, AM et al, 2013). PLA is a semi-crystalline exists as sterio
58
isomers i.e. poly (l-lactic acid), PLLA and poly (d-lactic acid), PLDA conventionally. It is
59
thermoplastic with good mechanical strength and, slightly soluble in water. PLA is derived
60
from renewable resources and is used in the automobile and packaging industries,
61
(Auras, et al, 2004). Polylactic acid (PLA) has been widely used as the hydrophobic
62
moieties to form the amphiphilic copolymers for drug delivery (Jiang, et al 2009; Kohori et
63
al., 1998; Kwon and Forrest, 2006), owing to its good mechanical property,
64
biodegradability and non-toxic degradation products to the living organisms (Tsuji, 2005).
Ac ce p
te
d
M
an
us
cr
ip t
42
3
Page 3 of 30
Numerous studies reported that the core which generally consists of a biodegradable
66
polymer such as poly (D, L-lactic acid) (PDLLA), copolymers and micelles. Grafting of
67
xyloglucan (XG) with L-lactide was investigated for potential use in cellulose fiber-
68
reinforced l-lac composite applications (Andrew Maraisa et al, 2012). This study prepares
69
new polymeric micelles by grafting of PLA on xyloglucan. A new amphiphilic co-polymer
70
was synthesized by grafting of xyloglucan with PLA by ring opening polymerization
71
reaction. Structural properties of this copolymer were characterized by NMR, FTIR. The
72
micelles of synthesized copolymer then prepared for the delivery of curcumin. The
73
properties of prepared micelles were further studied through photon correlation
74
spectroscopy and scanning electron microscopy. The unique properties of the micelles
75
are demonstrated by investigating the delivery behaviors of curcumin, a model drug.
76
2. Experimental
77
2.1 Materials
78
Xyloglucan (less than 47% galactose removal ratio) was obtained from DSP Gokyo Food
79
and Chemical Co. Ltd. (Fukusima, Japan). L-lactide was purchased from Sigma Aldrich
80
India. Curcumin was purchased from Himedia Lab Pvt. Ltd. (Product no.155108).
81
Methanol, benzyl alcohol and stannous octoate (Sn (Oct)2), Acetonitrile HPLC grade
82
solvent were obtained from chemical supplier and used as received.
83
2.2 Synthesis of grafted copolymer
84
The copolymerization of l-lactide and XG was carried out by ring opening polymerization,
85
briefly XG (0.5 g) and recrystallized l-lactide (25 g) were dissolved into a flask containing
86
dry toluene (about 40 mL). Benzyl alcohol was added and the flask was then sealed with
Ac ce p
te
d
M
an
us
cr
ip t
65
4
Page 4 of 30
a rubber septum and degassed by three argon vacuum cycles. The mixture was heated to
88
110°C with constant stirring, the catalyst, Sn (Oct)2, was added (0.4 mL) was added to
89
above mixture. The polymerization reaction was continued for about 2 h. (Andrew
90
Maraisa et al, 2012). The modified polymer sample has been separated by filtration then
91
dried and yield was calculated.
92
2.3. Characterization of graft copolymer
93
2.3.1. FTIR spectroscopy
94
FTIR characterization was carried on a spectrometer (Shimadzu 8400S, Japan). The
95
samples were triturated uniformly mixed with potassium bromide to get transparent mass
96
at 1:100 (drug: KBr) ratio. The KBr discs were compressed, under force of 15 tones for 3
97
to 4 min in a KBr pellet press (Kimaya engineers, India).
98
2.3.2. NMR spectroscopy
99
NMR spectroscopy of xyloglucan and grafted xyloglucan was performed using NMR
100
spectrometer (Bruker Avance III, 400 MHz) with following conditions of measurement,
101
superconducting Magnet (Magnet 9.4 T); 400 MHz probe, with Z-gradient, and 2H lock;
102
for observation of nuclei like 1H, 13C, 31P, 15N, etc. with 1H decoupling. The xyloglucan
103
and xyloglucan graft as powder were scanned between 1 to 10 ppm.
104
2.3.3 In vitro degradation in simulated fluids
105
The grafted polymer film samples for weight loss t were squared with about 0.05–0.1 mm
106
thickness and 2 cm length of side (Suggs, LJ, et. al 1998). The samples were immersed
Ac ce p
te
d
M
an
us
cr
ip t
87
5
Page 5 of 30
in SBF which content in small vials. The SBF solution was buffered at pH 7.4 with tris
108
(hydroxyl methyl) amino methane and hydrochloric acid.
109
% Weight loss
110
The weight loss w as calculated by comparing the dry weight (Wd) of the remained
111
sample after degradation for a predetermined time with the original dry weight (W0) of the
112
sample as the equation (Shi, R, et.al, 2010). At pre-determined intervals of 0, 4, 8, 12, 16,
113
20, 24, 28, 32 & 36 min; samples were taken out, purged with distilled water and
114
subsequently dried until absolute desiccation, then weighted.
an
us
cr
ip t
107
M
------------- (1)
115
2.3.4 In vivo Biodegradation study
117
For the study of in-vivo study 3 male wistar rats are selected to monitor the in vivo
118
degradation, grafted polymer films were subcutaneously implanted on the backs of male
119
wistar rats (200-300 g). Anesthesia was induced by intra-peritoneal injection of a mixture
120
of ketamine HCl (85 mg/kg body weight) and xylazine (12 mg/kg body weight)
121
(Gogolewski, S, et al. 1993). Tetracycline, 10mg/kg dose, was given at the time of
122
surgery. An incision (2.5 cm) was inflicted laterally about the mid portion of the back.
123
Subcutaneous pockets were formed around each incision, free film was inserted, and the
124
wounds were closed by intermittent nylon sutures, 0.5 cm apart for 3 individual male
125
wistar rats (Satturwar PM, et al.2003). Films were explanted at 10, 20, and 30 minutes for
126
analysis.
Ac ce p
te
d
116
127 6
Page 6 of 30
128
2.4 Determination of CMC and preparation of drug loaded polymeric micelles
130
The critical micelle concentration (CMC) is determined using a stalagnometer by
131
measuring the surface tension of a series of concentrations. Surface tension is most
132
conveniently calculated by the below formula.
(2)
us
----------------------------
cr
σ=σ0·n0/n
133
ip t
129
Where σ0 - surface tension of solvent (i.e. of water at t = 25o C, is equal to 72.75·10-3
135
N/m); n and n0 - numbers of drops of solvent and polymeric solution.
136
The surface tension is linearly dependent on the logarithm of concentration over a wide
137
range and is extensively independent of the concentration above the CMC. The CMC
138
obtained from the intersection between the regressions the straight lines of the linearly
139
dependent region and line passing through the plateau(Bhal Arun et al, 2011).
140
Curcumin loaded Micelles of PLA grafted xyloglucan copolymer was prepared by a single
141
step direct dissolution process briefly, 15 mg of curcumin and 150 mg PLA-grafted
142
xyloglucan with varying ratios were dissolved in water under mild stirring. Then, curcumin
143
was uniformly distributed in copolymer. The curcumin micelle solution was filtered through
144
a 0.22 mm syringe filter and was freeze dried and stored at 4° C prior to use.
145
2.5 Characterization of curcumin loaded PLA-grafted xyloglucan micelles
146
2.5.1 Size distribution of polymeric micelles
Ac ce p
te
d
M
an
134
7
Page 7 of 30
The particle size distribution and poly dispersity index (PDI) was determined using
148
Malvern Zetasizer (ZS 90, Malvern ltd., UK). The measurement is based on the dynamic
149
light scattering (DLS) phenomena in which the statistical intensity fluctuations of the
150
scattered light from the particles in the measuring cell are measured. Prior to the analysis,
151
sample was diluted with distilled water to generate appropriate scattering intensity. The
152
MPS and PDI values were obtained at an angle of 90° using disposable polystyrene cells
153
having 10 mm diameter cells at 25°C, which were equilibrating for 120 seconds.
154
2.5.2 Zeta potential determination
155
Zeta potentials of the micelles were determined using Zeta sizer (model, ZS 90, Malvern,
156
UK). Polymeric micelles were suspended in distilled water to obtain stock concentration of
157
1% (w/w) at 25°C. This polymeric micelle suspension was filled in cell with two electrodes
158
and zeta potential was determined.
159
2.5.3 Morphological examination
160
The morphology of micelles was studied by scanning electron microscopy (JSM 6390,
161
Japan). Samples of micelles were dusted on an aluminum stub and coated with gold
162
using a cold sputter coater to a thickness of 400°A, and then imaged using a 20 Kv
163
electron beam.
164
2.5.4 Drug loading and entrapment efficiency
165
The amount of curcumin in the micelles was determined with UV-Vis spectrophotometer
166
(Model No. UV-1700, Shimadzu, Japan) at 423 nm. Prior to determination the micelles
167
were suitably diluted with alcohol. The percent drug-loading (DL %) and percent
Ac ce p
te
d
M
an
us
cr
ip t
147
8
Page 8 of 30
168
entrapment efficiency (EE %) of Curcumin in prepared micelles was calculated using the
169
following equations (Jansson, 2004).
170
% EE
171
amount of drug in micelles x100 total mass of drug loaded micelles
---- (3)
us
cr
ip t
=
_____ (4)
an
172
2.5.5 In vitro release study
174
The release of curcumin from the micelles was investigated by dialysis method. The in
175
vitro release of curcumin was achieved using PBS (pH 7.4) as the release medium.
176
µl of curcumin loaded micelles or curcumin solution (in propylene glycol) as control were
177
introduced in a dialysis membrane bag (Mol wt. cut-off 12000 Da). The bag was
178
suspended in 200 ml of phosphate buffer (pH 7.4) while being stirred maintained at 37 °C.
179
To assure the adequate sink condition; the whole release medium was replaced by a
180
fresh medium. The curcumin concentration in PBS was determined by UV
181
spectrophotometer, at wavelength of 423 nm (Ali Fattahi, 2012).
182
Drug release mechanism
183
In order to study drug release mechanism from curcumin loaded polymeric micelle, the
184
drug release data was fitted in Korsmeyer-Peppas equation. The logarithmic plot of
185
cumulative percentage of drug released vs. log time gives the release exponent n and K
186
value from the slope of the straight line and y intercept respectively. (Peppas, 1985).
200
Ac ce p
te
d
M
173
9
Page 9 of 30
187
Mt / M∞ = Ktn
188
2.6 In vitro assessment of developed dry powder inhaler
189
Freeze dried powder was subjected to Andersen cascade impactor (ACI, SS 316, Copley
190
Scientific Ltd.) studies to determine mass median aerodynamic diameter (MMAD) and
191
geometric standard deviation (GSD) (De Boer AH, et al. 2003; Jensen, et. al.2010; Muttil,
192
P, 2007). ACI utilizes eight jet stages make possible classification of aerosols and allows
193
airborne particulates to impact upon stainless steel impaction surfaces. Starting at the
194
filter, a cumulative mass deposition (under size in percentage) vs. cut-off diameter of
195
respective stages was derived, the mean mass aerodynamic diameter (MMAD),
196
geometric standard diameter (GSD), fine particle dose (FPD), % fine particle fraction
197
(%FPF) and % mass balance was calculated (Mahajan HS, Gundare SA, 2014).
198
2.7 In vivo studies:
199
All animal experiments were approved and performed in accordance with the guidelines
200
of by Institutional Animal Ethics Committee of R.C. Patel Institute of Pharmaceutical
201
Education and Research, Shirpur, resolution number RCPIPER/IAEC/2014-15/22. Male
202
Wistar rats of 6-8 weeks old weighing 250–270 g were selected for the in vivo analysis
203
studies which were divided into two group, one for intratracheal administration of the
204
formulation and another for intratracheal administration of the curcumin suspension.
205
a) Dose Administration:
206
The rats were anesthetized with an intraperitoneal injection of urethane (1 g/kg, 25% w/v)
207
and kept on a heating pad to maintain the body temperature. For intratracheal
208
administration anesthetized rats were placed on and trachea was exposed by blunt
Ac ce p
te
d
M
an
us
cr
ip t
------------------------------ (5)
10
Page 10 of 30
209
dissection sternohyoideus muscle and small midline incision was made over the trachea
210
between the fifth and sixth tracheal rings using a
211
cannulated with PEG 200 tubing (5-7cm) with the tip positioned approximately at the
212
bifurcation of trachea. Formulation (dose equivalent to 100mg/kg) were administered to
213
the Group 1 by inserting PEG tubing into, cannula which is attached with the micro
214
syringe with16 gauge needle. The powder was placed at the tip of needle and made 4-5
215
actuations of air pump to ensure the complete delivery of dose. Blood samples were
216
collected from tail vein intra-venously at 0.30, 1, 2, 4, 8, 16, 24, hrs. The rats were held at
217
45° position during intratracheal administration. To the Group 2 of curcumin suspension
218
(100mg/kg) was administered intratracheally by the same procedure mentioned above.
219
After administration the rats were sacrificed humanely at different time intervals and then
220
lungs were harvested by surgical resection. Both lung lobe was quickly rinsed with saline
221
and blotted up with filter paper to get rid of blood-taint and macroscopic blood vessels as
222
much as possible and weighed. Lungs were stored for up to 48 h in a deep freezer
223
(−70°C) until HPLC analysis (Misra et al., 2007, Beinborn et al., 2012).
224
b) Calibration curves of curcumin in lungs homogenate:
225
Calibration curves of curcumin were prepared with lung tissue homogenate by spiking the
226
known amounts of the drug utilizing its HPLC peak area ratio to the internal standard (4-
227
hydroxybenzophenone). The linear range of curcumin was 1–1000 ng/g for lung tissue.
228
The detection limits were 5ng/g for lung tissue samples.
229
C) Processing of samples:
230
i) Lung tissue:
Ac ce p
te
d
M
an
us
cr
ip t
20 guage needle followed by
11
Page 11 of 30
The lung tissue samples were homogenized with 1% w/w of saline in a tissue
232
homogenizer (Teflon homogenizer). To a 200 µlof lung homogenate, 50µl of the internal
233
standard (4-hydroxybenzophenone, 10 µg/mL) was spiked and vortex mixed for 30 s.
234
Then 0.5 ml of acetonitrile was added and vortex-mixed for 1 min. The sample was
235
centrifuged at 10000 rpm for 6 min at 4º C in ultracentrifuge (Beckman Coulter),
236
(Mukherjee et al., 2013). The supernatant layer (0.75 ml) was transferred to a 15 ml glass
237
test tube, and then 4.5 ml of extraction solvent, methyl t-butyl ether were added. The
238
sample was vortex-mixed for 3 min using a multi-tube vortex mixer and evaporated to
239
dryness under a stream of nitrogen at room temperature. 100 µl of supernant was
240
injected into the HPLC system after reconstitution.
241
ii) Chromatographic conditions:
242
The HPLC system (Agilent 1200 Series) consisted of C18 column (Eclipsed XDB 5 µm,
243
4.6 mm x 150 mm, Singapore), Ezchrome Elite Software, quaternary pump, Model G1354
244
A and Ultraviolet variable wavelength Diode Array detector, Model G1315D.
245
detection wavelength was 423 nm. The mobile phase was composed of acetonitrile–Citric
246
acid buffer (pH 3.0) (55:45) Elution was performed isocratically at 40°C at a flow rate of
247
1.0 mL/min (Zhimei Song, 2011).
248
iii) Pharmacokinetic analysis
249
The maximum plasma concentration (Cmax) and time of its occurrence (Tmax) were directly
250
computed from the plasma concentration v/s time plot. The Cmax, Tmax, and AUC were
251
computed using Kinetica 5® (Thermo Fisher Scientific) software. Relative bioavailability
252
(F) was calculated with reference to curcumin suspension.
The
Ac ce p
te
d
M
an
us
cr
ip t
231
12
Page 12 of 30
3.0 Results and discussion
254
3.1 Synthesis of grafted copolymer
255
Ring opening polymerization (ROP) reaction of L-lactide, xyloglucan and benzyl alcohol is
256
shown in Fig. 1. The graft copolymerization reaction has been carried out at in presence
257
a sacrificial initiator (benzyl alcohol), as the amount of initiating hydroxyl groups in
258
xyloglucan is uncertain. This co-initiator system has proved as an efficient method for ring
259
opening polymerization of lactides (Kricheldorf & Damrau, 1997; Lonnberg et al., 2006;
260
Trollsas et al., 2000). The time and temperature were kept constant during reaction.
261
Anionic ring-opening polymerization of cyclic ester monomers results of nucleophilic
262
attack of a negatively charged initiator on the carbonyl carbon or on the carbon atom
263
adjacent to the acyl-oxygen, resulting in linear polyester. The ROP reaction shows
264
formation of ester bond so it demonstrates that grafting of polymer was done successfully
265
(Andrew Maraisa et al, 2012).
266
3.2. Characterization of graft copolymer
267
3.2.1 IR spectra of xyloglucan and L-lactide grafted xyloglucan
268
The IR spectra of samples in KBr pellets were recorded using a FTIR spectrometer
269
(Shimadzu 8400S, Japan) in the range 450–4000 cm−1. FTIR spectra for xyloglucan and
270
grafted xyloglucan samples are shown in Fig. 2. The grafting of L-lactide chains from the
271
xyloglucan can evident in the FTIR spectra through the presence of new peak, around
272
1759 cm−1 in the carbonyl region.
273
3.2.2 NMR spectroscopy
Ac ce p
te
d
M
an
us
cr
ip t
253
13
Page 13 of 30
274
To confirm possible grafting of xyloglucan, 1H NMR spectra of xyloglucan and PLA
275
grafted xyloglucan was compared (Fig. 3). As illustrated in Fig. 3, the major chemical shift
276
of xyloglucan and grafted xyloglucan observed between 1 and 8 ppm. NMR spectra of
277
xyloglucan showed singlet at 2.1 (CH2) and multiplets at 3.91
278
OH), (O CH), 5.1 (O) and multiplets at 3.42, 3.76, due to CH (OH). NMR spectra of
279
modified polymer showed multiplets in the region 1.45 to 1.56
280
decreases intensity from 2.30 to 1.57 in the region of 3.57 to 3.74 proves successful
281
grafting of xyloglucan with poly lactic acid. (Silverstein et al, 2005).
282
3.2.3 In vitro Degradation
283
The weight remaining after degradation in-vitro was 62.85%. This phenomenon
284
demonstrated that the degradation and dissolution rate of grafted polymer was rapid SSF
285
in simulated body fluids under the normal body temperature.
286
3.2.4 In vivo degradation
ip t
(O CH2), 4.50 (O CH2
Ac ce p
te
d
M
an
us
cr
(OCO-CH3) and
287
After in vivo implantation in rats, the films showed weight loss of 90.23% with in 36
288
minutes. The bulk degradation is evident both in vitro and in vivo. Although the xyloglucan
289
and polylactic aciad are highly biodegradable polymer which presumably will lead to new
290
application of these polymers in the field of novel drug delivery system.
291
3.3 Determination of CMC and preparation of drug loaded polymeric micelles
292
As the structure of the grafted copolymers consists of hydrophobic and hydrophilic blocks,
293
hence the ability of micelle formation as reservoirs for drugs is of interest. The surface
294
tension of 0.1% solution polymer solution, measured by conventional stalagmometry
14
Page 14 of 30
method (number of drops of liquids between marks), has found to be 99.48 *10-3 N/m. To
296
calculate CMC from each curves two tangents were drawn to the point near the place
297
where the curves bended. The extrapolation of the crossing point of these tangents to the
298
abscissa axis gives the value of CMC. The critical micelle concentration of the grafted
299
xyloglucan micelle suspension was 1.5 X 10-2 % w/w.
300
3.4 Characterization of Curcumin loaded PLA-grafted xyloglucan micelles
301
3.4.1 Size distribution of polymeric micelles
us
cr
ip t
295
The mean particle size of the curcumin loaded micelles measured using the particle size
303
analyzer was 102.4 nm, with polydispersity index (PDI) of 0.275, signifying that the
304
narrow distribution of sizes of micelles. It is reported that the particle size will directly
305
influence carrier circulation time and distribution in vivo (Sezgin, et al. 2006). Small
306
particle sizes (<200 nm) could reduce the uptake of the reticulo endothelial system (RES)
307
and provide efficiently passive tumor-targeting ability via the enhanced permeability and
308
retention (EPR) effects (Dabholkar.et al 2006). Therefore, size of prepared curcumin
309
micelles was suitable for passive tumor targeting.
310
3.4.2 Zeta potential measurements
311
The zeta potential represents electrical charge to the micelle surface. It is important and
312
useful parameter to predict and control the stability of colloidal dispersion. The
313
measurement of zeta potential is an important tool to understand the dispersion
314
phenomenon. The zeta potential of pure drug, blank micelles and drug loaded micelles
315
were 9.69 mV, -21.7mV and -18.2 mV respectively. The greater the value, more likely the
316
colloidal system is to be stable because the charged particles repel one another and thus
Ac ce p
te
d
M
an
302
15
Page 15 of 30
overcome the natural tendency to aggregate. It is recognized that higher zeta potential
318
values, either positively or negatively charged, demonstrates that dispersion will have
319
greater long-term stability.
320
3.4.3 Morphological examination
321
Representative SEM image obtained for curcumin loaded polymeric micelles is shown in
322
Fig.4. The shape of dried micelles was mainly spherical and their size agreed well with
323
that measured by dynamic light scattering (DLS).
324
3.4.4 Drug loading and entrapment efficiency
325
Entrapment of hydrophobic drug into micelles was results of chemical conjugation or
326
physical entrapment. During physical entrapment of drug in micelles various forces
327
involved including hydrogen bond and van-der Waals forces. Curcumin was encapsulated
328
into Xyloglucan grafted L-lactide micelles with loading capacity of 68.9 ± 0.02% and
329
entrapment efficiency of 96.38± 0.2% respectively. The lower drug loading was attributed
330
to limited aqueous solubility of grafted copolymer.
331
3.4.5 In vitro drug release study
332
In vitro release of curcumin from micelle formulation under sink condition was investigated
333
by dialysis method PBS (pH 7.4) release medium. As shown in Fig. 5A, at the end of 5 h
334
about 50% of curcumin released from polymeric micelles, where as about 75% of
335
curcumin was released from the propylene glycol solution. The results showed that
336
curcumin follows a sustained-release property from polymeric micelle that was similar to
337
previously reported studies. The slope of the plot (Fig 5B) was 0.81 demonstrating the
Ac ce p
te
d
M
an
us
cr
ip t
317
16
Page 16 of 30
anomalous (non fickian) release kinetic i.e. release of curcumin from micelles followed
339
erosion- diffusion mechanism (Peppas NA, Shalin JJ, et al 1989).
340
In vitro assessment of dry powder inhaler
341
The freeze dried powder micelles were subjected for the in vitro performance and
342
aerodynamic property using Anderson cascade impactor. The data obtained for various
343
aerodynamic properties demonstrates that 0.4% drug retained in the capsule and device
344
that has given indication of good dispersibility of formulated DPI and about 99.6% of drug
345
emitted from DPI. Fine particle fraction (FPF) was found to be 12.16%. The Mass Median
346
Aerodynamic Diameter (MMAD) of the curcumin loaded micelles was 106.67nm and was
347
less than 5μm which is prerequisite to have an inhalation of powders into the lower region
348
of the lung. The geometric standard diameter (GSD) found to be 68.77 nm, which was
349
depending on aerodynamic behavior.
350
In vivo studies
351
The in vivo studies conducted in this investigation to test the possibility of assessing
352
targeting ability of novel grafted polymer micelles to lungs. The drug distribution
353
parameters in lungs were calculated and are represented Table 1. The Cmax was
354
50342±1088.8 ng/ml at Tmax of 180 min for curcumin suspension and 89301±8182.1
355
ng/ml at Tmax of 120 min for powder micelle formulation respectively. Eventually, there
356
was an increase in AUC0-480
357
suspension thereby confirms the maintenance of effective drug concentration in lung
358
tissue for prolonged period as compared to suspension due to the ability of micelles in
359
reaching the pulmonary region. The relative bioavailability of curcumin when administered
360
as micelles formulation was about 539 ± 9.86, reflecting the potential use of micelles as
Ac ce p
te
d
M
an
us
cr
ip t
338
min
for DPIs compared with the AUC0-480min of curcumin
17
Page 17 of 30
novel carrier for the poorly bioavailable curcumin.
362
4.0 Conclusion
363
Newly synthesized L-lactide grafted xyloglucan copolymer can self-assemble to form
364
micelles with 0.0150 wt% CMC. Average particle size was 102.4 nm with 0.275 PDI. The
365
polymeric micelles, exhibited higher entrapment efficiency and drug loading for curcumin.
366
In vitro dry powder for inhalation performance observed for developed freeze dried
367
micelles suggesting high deep lung deposition of drug. The data of in vivo studies showed
368
high and prolonged residence of curcumin within lungs after pulmonary administration.
369
Novel grafted micelles expected to provide improved local concentration. The results of
370
present study clearly indicated promising potential of novel grafted polymer micelles for
371
delivering curcumin to deep lungs for effective treatment of lungs cancer.
372
Acknowledgement
373
Contributors are grateful to DSP Gokyo Food and Chemical Ltd. (Fukusima, Japan) for
374
gifting xyloglucan and Dr.S.J.Surana (Principal R.C.Patel IPER Shirpur) for providing
375
essential facilitates required carrying out research work.
376
5.0 References
Ac ce p
te
d
M
an
us
cr
ip t
361
377
Ali Fattahia, Mohammad-Ali Golozara, Jaleh Varshosazb, Hamid Mirmohammad
378
Sadeghid, Mohammadhossein Fathia. (2012).Preparation and characterization of
379
micelles of oligomeric chitosan linked to all-trans retinoic acid, Carbohydrate
380
Polymers. 87, 1176– 1184.
18
Page 18 of 30
Andrew Maraisa, Joby J. Kochumalayil., (2012). Toward an alternative compatibiliser
382
for PLA cellulose composites: Grafting Of xyloglucan with PLA, Carbohydrates
383
polymer, 89, 1038-1043.
384
Auras, R., Harte, B., & Selke, S. (2004). An overview of polylactides as packaging
385
materials. Macromolecular Bioscience, 4(9), 835–864.
386
Avachat, AM., Gujar, KN., Wagh, KV. (2013).Development and evaluation of tamarind
387
seed
388
Carbohydrate Polymers, 91, 537–542.
389
Bahl AB, Bahl BS, Tuli GD.(2011) Essentials of Physical Chemistry ,Multicolor Edition
390
S Chand Publishers.428-429..
391
Beinborn, NA, Wiederhold, NP, Hugh, DC and Williams (2012). Dry powder
392
insufflation of crystalline and amorphous voriconazole formulations produced by thin
393
film freezing to mice. European Journal of Pharmaceutics and Biopharmaceutics, 81,
394
1, 600–608.
395
Buckeridge, MS., Santos, HP, Tine, M.A.S. (2003). Mobilization of storage cell wall
396
polysaccharides in seeds. Plant Physiology and Biochemistry, 8(1–2), 141–156.
397
Carpita, NC. , Gibeaut, DM. (1993). Structural models of primary cell walls in flowering
398
plants: Consistency of molecular structure with the physical properties of the walls
399
during growth. Plant Journal, 3(1), 1–30.
400
Dabholkar, RD., Sawant, RM., Mongayt, DA, Devarajan, PV, Torchilin, VP. (2006).
401
Polyethylene glycol-phosphatidyl ethanolamine conjugate (PEG-PE)-based mixed
buccal
films
of
rizatriptan
benzoate.
M
an
mucoadhesive
Ac ce p
te
d
xyloglucan-based
us
cr
ip t
381
19
Page 19 of 30
micelles: some properties, loading with paclitaxel, and modulation of P-glycoprotein-
403
mediated efflux, International Journal of Pharmaceutics. 315, 148–157.
404
Gogolewski, S, Jovanovic, M, Perren, SM, Dillon, JG, Hughes, MK. (1993). Tissue
405
response and in vivo degradation of selected polyhydroxyacids: polylactides (PLA),
406
poly (3-hydroxybutyrate) (PHB) and poly (3-hydroxybutyrate-cp-3-hydroxyvalerate)
407
(PHB/VA).Journal of Biomedical Material Research.27, 1135-1148.
408
Jansson, J. Schille, K. Olofsson, G. Cardoso, R. Loh, W. (2004). The interaction
409
between PEO–PPO–PEO triblock copolymers and ionic surfactants in aqueous
410
solution studied using light scattering and calorimetry, Journal of Physical Chemistry.
411
B 108, 82–92.
412
Jiang, M., Wu, Y., He, Y., Nie, J. (2009). Micelles formed by self-assembly of hyper-
413
branched poly [(amine-ester)-co-(d,l-lactide)] (HPAE-co-PLA) copolymers for protein
414
drug delivery. Polymer International, 58(1), 31–39.
415
Kohori, F., Sakai, K., Aoyagi, T., Yokoyama, M., Sakurai, Y., Okano, T.
416
(1998).Preparation and characterization of thermally responsive block copolymer
417
micelles comprising poly (N-isopropyl acrylamide-b-dl-lactide). Journal of Controlled
418
Release, 55(1), 87–98.
419
Kricheldorf, HR., & Damrau, DO. (1997). Polylactones. 37. Polymerizations of l-lactide
420
initiated with Zn (II) l-lactate and other resorbable Zn salts. Macromolecular Chemistry
421
and Physics, 198(6), 1753–1766.
422
Kumar R., Srivastava A., Behari, K. (2009). Synthesis and characterization of
423
polysaccharide
Ac ce p
te
d
M
an
us
cr
ip t
402
based
graft
copolymer
by
using
potassium
peroxymonosul-
20
Page 20 of 30
phate/ascorbic acid as an efficient redox initiator in inert atmosphere. Journal of
425
Applied Polymer Science, 112(3), 1407–1415.
426
Kwon, GS. Forrest, ML. (2006). Amphiphilic block copolymer micelles for nanoscale
427
drug delivery. Drug Development Research, 67(1), 15–22.
428
Letchford, K., Burt, H. (2007). A review of the formation and classification of
429
amphiphilic block copolymer nanoparticulate structures: Micelles, nanospheres,
430
nanocapsules and polymersomes. European Journal of Pharmaceutics and
431
Biopharmaceutics, 65(3), 259–269.
432
Lonnberg, H., Zhou, Q., Brumer, H., Teeri, T.T., Malmstrom, E., Hult, A. (2006).
433
Grafting of cellulose fibers with poly (epsilon-caprolactone) and poly(l-lactic acid) via
434
ring-opening polymerization. Biomacromolecules, 7(7), 2178–2185.
435
Maeda, T., Yamashita, H., Morita, N. (2007). Application of xyloglucan to improve the
436
gluten membrane on bread making. Carbohydrate Polymers, 68, 658–664.
437
Mahajan HS. Gundare SA. (2014). Preparation, characterization and pulmonary
438
pharmacokinetics of xyloglucan microspheres as dry powder inhalation. Carbohydrate
439
Polymers.102, 529–536.
440
Mishra, MM. Tripathy, J, Yadav, M., Sand, A., & Behari, K. (2010). Graft copolymer (K-
441
carrageenan-g-N-vinyl formamide): Preparation, characterization and application.
442
Carbohydrate Polymers, 80, 235–241,
Ac ce p
te
d
M
an
us
cr
ip t
424
21
Page 21 of 30
443
Misra, A, Chougule, M, Padhi, B. (2007). Nano-liposomal dry powder inhaler of
444
tacrolimus:
445
International Journal of Nanomedicine, 2, 4, 675–688.
446
Mukherjee, B, Sinha, B, Pattnaik, G (2013). Poly-lactide-co-glycolide nanoparticles
447
containing voriconazole for pulmonary delivery: in vitro and in vivo study, Nanomed:
448
Nanotech, Bio, Med, 9, 1, 94–104.
449
Peppas NA, Shalin, J.J. (1989). A simple equation for the description of solute release
450
III. Coupling of diffusion and relaxation. International Journal of Pharmaceutics, 57,
451
169-172.
452
Peppas, NA (1985), Analysis of fickian and non fickian drug release from polymers’,
453
Pharm ActaHelv, vol. 60, 110-111.
454
Rao, PS., Srivastava, HC. (1973). Industrial gums: Polysaccharides and their
455
derivatives. Diego: Academic Press. 369–411.
456
Satturwar, PM, Fulzele, SV, Dorle, AK. (2003) Biodegradation and In vivo
457
biocompatibility of rosin: a natural film-forming polymer. AAPS PharmSciTech 4, 1-7.
458
Sezgin, Z.
459
polymeric micelles for solubilization of poorly soluble anticancer drugs, European
460
Journal of Pharmaceutics and Biopharmaceutics. 64, 261–268.
461
Shi, R, Zhu, A, Chen, D., (2010) In vitro degradation of starch/PVA films and
462
biocompatibility evaluation. Journal of Applied Polymer Science, 115, 346-357.
characterization,
and
pulmonary
Pharmacokinetics,
Ac ce p
te
d
M
an
us
cr
ip t
Preparation,
Yuksel, N. Baykara, T. (2006). Preparation and characterization of
22
Page 22 of 30
Silverstein, RM., Webster, FX., Kiemle, DJ. (2005). Spectrophotometric identification
464
of organic compounds. John Wiley and Sons, 150–151.
465
Suggs, LJ, Krishnan, RS, Garcia, CA, Peter, SJ, Anderson, JM, Mikos, AG. (1998) In
466
vitro and in vivo degradation of poly(propylene fumarate co-ethylene glycol) hydrogels.
467
Journal Biomedical material Research, 42, 312-320.
468
Tripathy, J., Mishra, D. K., Yadav, M., Sand, A., Behari, K. (2009). Modification of -
469
carrageenan by graft copolymerization of methacrylic acid: Synthesis and applications.
470
Journal of Applied Polymer Science, 114(3), 3896–3905.
471
Trollsas, M., Claesson, H., Atthoff, B., Hedrick, JL, Pople, JA, Gast, AP. (2000).
472
Conformational and structural properties of high functionality dendrimer-like star
473
polymers
474
Symposia, 153, 87–108.
475
Tsuji, H. (2005). Poly (lactide) stereo complexes: Formation, structure, properties,
476
degradation, and applications. Macromolecular Bioscience, 5(7), 569–597.
477
Zhimei Song, (2011). Curcumin-loaded PLGA-PEG-PLGA triblock copolymeric
478
micelles: Preparation, pharmacokinetics and distribution in vivo, Journal of Colloid and
479
Interface Science, vol. 354, 116–123.
living polymerization techniques. Macromolecular
d
from
Ac ce p
te
synthesized
M
an
us
cr
ip t
463
480 481
Figure legends
482
Fig 1: Scheme of grafting of xyloglucan with L-lactide by Ring Opening Polymerization
483
(ROP)
484
Fig 2: FTIR spectra of native (A) and grafted xyloglucan (B) 23
Page 23 of 30
485
Fig 3: NMR spectra of xyloglucan (bottom) and grafted xyloglucan (top)
486
Fig 4: Scanning electron microscopic images of polymeric micelles
487
Fig 5: In vitro drug release from polymeric micelles (A), drug release mechanism (B).
ip t
488 489
Table 1: Pharmacokinetic parameters following intratracheal administration curcumin
491
micelles as dry powder inhaler formulation and suspension. Suspension 50342 ± 1088.8
an
Cmax (ng/ml) Tmax (min)
89301± 8182.1 120
24132010±90183.4
53241082±403215.0
--
539 ± 9.86
492 493
Ac ce p
HIGHLIGHTS
te
d
Relative bioavailability
Micelles as DPI
180
M
AUC 0-480 min (ng/ml*min)
494
us
Pharmacokinetic Parameter
cr
490
495
PLA grafted xyloglucan copolymer was synthesized successfully by ring opening
496
polymerization.
497
IR and NMR analysis results reveal successful grafting of PLA with xyloglucan.
498
Produced micelle showed suitable particles size, CMC, drug loading and release
499
efficiency.
500
PLA grafted xyloglucan based micelles are promising drug carriers.
501 502 24
Page 24 of 30
503 504
te
d
M
an
us
cr
ip t
Ac ce p
505
25
Page 25 of 30
Ac
ce
pt
ed
M
an
us
cr
i
Figure(s)
Page 26 of 30
Ac
ce
pt
ed
M
an
us
cr
i
Figure(s)
Page 27 of 30
Ac ce p
te
d
M
an
us
cr
ip t
Figure 3
Page 28 of 30
Ac
ce
pt
ed
M
an
us
cr
i
Figure 4
Page 29 of 30
Ac
ce
pt
ed
M
an
us
cr
i
Figure(s)
Page 30 of 30
S0141-8130(15)00668-6 http://dx.doi.org/doi:10.1016/j.ijbiomac.2015.09.053 BIOMAC 5389
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
23-7-2015 28-8-2015 27-9-2015
Please cite this article as: H.S. Mahajan, P.R. Mahajan, Development of grafted xyloglucan micelles for pulmonary delivery of curcumin: In vitro and in vivo studies, International Journal of Biological Macromolecules (2015), http://dx.doi.org/10.1016/j.ijbiomac.2015.09.053 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.
Development of grafted xyloglucan micelles for pulmonary delivery of
2
curcumin: In vitro and in vivo studies
3
Hitendra S Mahajan*, Prashant R Mahajan
4
R.C. Patel Institute of Pharmaceutical Education and Research Shirpur, Dist Dhule.
cr
ip t
1
us
5 6
an
7
M
8
d
9
*corresponding author
11
Dr. Hitendra S Mahajan
12
R. C. Patel Institute of Pharmaceutical Education & Research,
13
Near Karvand Naka, Shirpur-425405,
14
Dist: Dhule, Maharashtra, India.
15
E mail- [email protected]
16
M.No – 91-9423487043,
17
Phone- 91-2563255189 (office)
18
Fax – 91-2563255180
Ac ce p
te
10
19 1
Page 1 of 30
Abstract
21
A novel grafted copolymer consisting of L-lactide grafted xyloglucan was synthesized by
22
polymerization reaction and characterized. The grafted copolymers were analyzed by
23
Fourier-transform infrared spectrometry (FT-IR) and 1H nuclear magnetic resonance (1H
24
NMR) was performed to confirm the grafting of L-poly lactic acid on xyloglucan. The
25
grafted polymer forms micelles at the critical micelle concentration of 0.0150 wt% with the
26
average particle size of 102 nm, as determined by particle size analyzer. The zeta
27
potential of the curcumin loaded micelles was -18.2 mV, an acceptable drug loading
28
efficiency of 68.9 ± 0.02% and the entrapment efficiency of 96.38± 0.2%. The release
29
study for 5h showed a sustained release property. In vitro assessment demonstrates
30
suitability of micelles as dry powder for inhalation. In vivo studies showed significant
31
improvement in bioavailability on pulmonary administration of curcumin micelles as DPI
32
formulation. The potential for pulmonary delivery curcumin loaded in micelles was
33
evaluated. In conclusion, polymeric micelle based on a newly synthesized grafted
34
xyloglucan could be suitable carrier for pulmonary delivery of curcumin.
35
Key words- Grafted polymer, xyloglucan, micelles, drug release, curcumin
36
1. Introduction
37
Naturally occurring carbohydrate polymers are obtained from the plant, animal and
38
microbial sources. Recently, much consideration has been given to chemical modification
39
of natural polymers (Kumar, et al 2009, Mishra, et al 2010; Tripathy, et al 2009).
40
Amphiphilic copolymers, composed of hydrophilic and hydrophobic moieties, can form
41
micelles spontaneously or other self-aggregates in an isotropic aqueous solution. These
Ac ce p
te
d
M
an
us
cr
ip t
20
2
Page 2 of 30
polymeric self-aggregates have been recognized as a potential delivery carrier as the
43
water insoluble core can serve as a reservoir for different hydrophobic agents. These
44
amphiphilic copolymers usually consist of biocompatible, biodegradable hydrophobic
45
polymer blocks such as polyesters or poly (amino acids) covalently bonded to a
46
biocompatible hydrophilic block such as polyethylene glycol or natural polysaccharides.
47
(Letchford and Burt, 2007).
48
Seed xyloglucan (XG) is a natural, water-soluble and non toxic polysaccharide. It has
49
cross-linked conformation and consists of a (1→4)-β-D-glucan backbone and (1→6)-α-D-
50
xylose branches that are partially substituted by (1→2)-β-D-galactoxylose (Carpita and
51
Gibeaut, 1993). Xyloglucan play important roles in the control of cell expansion in the
52
seeds of many dicotyledons (Buckeridge, et al 2003). These seed xyloglucans obtained
53
from seeds of Tamarindus indica had a numerous applications as common additives for
54
food and cosmetic, where they act as thickeners and stabilizing agents (Rao and
55
Srivastava, 1973, Maeda, et al, 2007). Xyloglucan has been studied as a potential drug
56
delivery carrier because it is non-toxic, non-mutagenic, non-irritant, blood compatible and
57
biodegradable (Avachat, AM et al, 2013). PLA is a semi-crystalline exists as sterio
58
isomers i.e. poly (l-lactic acid), PLLA and poly (d-lactic acid), PLDA conventionally. It is
59
thermoplastic with good mechanical strength and, slightly soluble in water. PLA is derived
60
from renewable resources and is used in the automobile and packaging industries,
61
(Auras, et al, 2004). Polylactic acid (PLA) has been widely used as the hydrophobic
62
moieties to form the amphiphilic copolymers for drug delivery (Jiang, et al 2009; Kohori et
63
al., 1998; Kwon and Forrest, 2006), owing to its good mechanical property,
64
biodegradability and non-toxic degradation products to the living organisms (Tsuji, 2005).
Ac ce p
te
d
M
an
us
cr
ip t
42
3
Page 3 of 30
Numerous studies reported that the core which generally consists of a biodegradable
66
polymer such as poly (D, L-lactic acid) (PDLLA), copolymers and micelles. Grafting of
67
xyloglucan (XG) with L-lactide was investigated for potential use in cellulose fiber-
68
reinforced l-lac composite applications (Andrew Maraisa et al, 2012). This study prepares
69
new polymeric micelles by grafting of PLA on xyloglucan. A new amphiphilic co-polymer
70
was synthesized by grafting of xyloglucan with PLA by ring opening polymerization
71
reaction. Structural properties of this copolymer were characterized by NMR, FTIR. The
72
micelles of synthesized copolymer then prepared for the delivery of curcumin. The
73
properties of prepared micelles were further studied through photon correlation
74
spectroscopy and scanning electron microscopy. The unique properties of the micelles
75
are demonstrated by investigating the delivery behaviors of curcumin, a model drug.
76
2. Experimental
77
2.1 Materials
78
Xyloglucan (less than 47% galactose removal ratio) was obtained from DSP Gokyo Food
79
and Chemical Co. Ltd. (Fukusima, Japan). L-lactide was purchased from Sigma Aldrich
80
India. Curcumin was purchased from Himedia Lab Pvt. Ltd. (Product no.155108).
81
Methanol, benzyl alcohol and stannous octoate (Sn (Oct)2), Acetonitrile HPLC grade
82
solvent were obtained from chemical supplier and used as received.
83
2.2 Synthesis of grafted copolymer
84
The copolymerization of l-lactide and XG was carried out by ring opening polymerization,
85
briefly XG (0.5 g) and recrystallized l-lactide (25 g) were dissolved into a flask containing
86
dry toluene (about 40 mL). Benzyl alcohol was added and the flask was then sealed with
Ac ce p
te
d
M
an
us
cr
ip t
65
4
Page 4 of 30
a rubber septum and degassed by three argon vacuum cycles. The mixture was heated to
88
110°C with constant stirring, the catalyst, Sn (Oct)2, was added (0.4 mL) was added to
89
above mixture. The polymerization reaction was continued for about 2 h. (Andrew
90
Maraisa et al, 2012). The modified polymer sample has been separated by filtration then
91
dried and yield was calculated.
92
2.3. Characterization of graft copolymer
93
2.3.1. FTIR spectroscopy
94
FTIR characterization was carried on a spectrometer (Shimadzu 8400S, Japan). The
95
samples were triturated uniformly mixed with potassium bromide to get transparent mass
96
at 1:100 (drug: KBr) ratio. The KBr discs were compressed, under force of 15 tones for 3
97
to 4 min in a KBr pellet press (Kimaya engineers, India).
98
2.3.2. NMR spectroscopy
99
NMR spectroscopy of xyloglucan and grafted xyloglucan was performed using NMR
100
spectrometer (Bruker Avance III, 400 MHz) with following conditions of measurement,
101
superconducting Magnet (Magnet 9.4 T); 400 MHz probe, with Z-gradient, and 2H lock;
102
for observation of nuclei like 1H, 13C, 31P, 15N, etc. with 1H decoupling. The xyloglucan
103
and xyloglucan graft as powder were scanned between 1 to 10 ppm.
104
2.3.3 In vitro degradation in simulated fluids
105
The grafted polymer film samples for weight loss t were squared with about 0.05–0.1 mm
106
thickness and 2 cm length of side (Suggs, LJ, et. al 1998). The samples were immersed
Ac ce p
te
d
M
an
us
cr
ip t
87
5
Page 5 of 30
in SBF which content in small vials. The SBF solution was buffered at pH 7.4 with tris
108
(hydroxyl methyl) amino methane and hydrochloric acid.
109
% Weight loss
110
The weight loss w as calculated by comparing the dry weight (Wd) of the remained
111
sample after degradation for a predetermined time with the original dry weight (W0) of the
112
sample as the equation (Shi, R, et.al, 2010). At pre-determined intervals of 0, 4, 8, 12, 16,
113
20, 24, 28, 32 & 36 min; samples were taken out, purged with distilled water and
114
subsequently dried until absolute desiccation, then weighted.
an
us
cr
ip t
107
M
------------- (1)
115
2.3.4 In vivo Biodegradation study
117
For the study of in-vivo study 3 male wistar rats are selected to monitor the in vivo
118
degradation, grafted polymer films were subcutaneously implanted on the backs of male
119
wistar rats (200-300 g). Anesthesia was induced by intra-peritoneal injection of a mixture
120
of ketamine HCl (85 mg/kg body weight) and xylazine (12 mg/kg body weight)
121
(Gogolewski, S, et al. 1993). Tetracycline, 10mg/kg dose, was given at the time of
122
surgery. An incision (2.5 cm) was inflicted laterally about the mid portion of the back.
123
Subcutaneous pockets were formed around each incision, free film was inserted, and the
124
wounds were closed by intermittent nylon sutures, 0.5 cm apart for 3 individual male
125
wistar rats (Satturwar PM, et al.2003). Films were explanted at 10, 20, and 30 minutes for
126
analysis.
Ac ce p
te
d
116
127 6
Page 6 of 30
128
2.4 Determination of CMC and preparation of drug loaded polymeric micelles
130
The critical micelle concentration (CMC) is determined using a stalagnometer by
131
measuring the surface tension of a series of concentrations. Surface tension is most
132
conveniently calculated by the below formula.
(2)
us
----------------------------
cr
σ=σ0·n0/n
133
ip t
129
Where σ0 - surface tension of solvent (i.e. of water at t = 25o C, is equal to 72.75·10-3
135
N/m); n and n0 - numbers of drops of solvent and polymeric solution.
136
The surface tension is linearly dependent on the logarithm of concentration over a wide
137
range and is extensively independent of the concentration above the CMC. The CMC
138
obtained from the intersection between the regressions the straight lines of the linearly
139
dependent region and line passing through the plateau(Bhal Arun et al, 2011).
140
Curcumin loaded Micelles of PLA grafted xyloglucan copolymer was prepared by a single
141
step direct dissolution process briefly, 15 mg of curcumin and 150 mg PLA-grafted
142
xyloglucan with varying ratios were dissolved in water under mild stirring. Then, curcumin
143
was uniformly distributed in copolymer. The curcumin micelle solution was filtered through
144
a 0.22 mm syringe filter and was freeze dried and stored at 4° C prior to use.
145
2.5 Characterization of curcumin loaded PLA-grafted xyloglucan micelles
146
2.5.1 Size distribution of polymeric micelles
Ac ce p
te
d
M
an
134
7
Page 7 of 30
The particle size distribution and poly dispersity index (PDI) was determined using
148
Malvern Zetasizer (ZS 90, Malvern ltd., UK). The measurement is based on the dynamic
149
light scattering (DLS) phenomena in which the statistical intensity fluctuations of the
150
scattered light from the particles in the measuring cell are measured. Prior to the analysis,
151
sample was diluted with distilled water to generate appropriate scattering intensity. The
152
MPS and PDI values were obtained at an angle of 90° using disposable polystyrene cells
153
having 10 mm diameter cells at 25°C, which were equilibrating for 120 seconds.
154
2.5.2 Zeta potential determination
155
Zeta potentials of the micelles were determined using Zeta sizer (model, ZS 90, Malvern,
156
UK). Polymeric micelles were suspended in distilled water to obtain stock concentration of
157
1% (w/w) at 25°C. This polymeric micelle suspension was filled in cell with two electrodes
158
and zeta potential was determined.
159
2.5.3 Morphological examination
160
The morphology of micelles was studied by scanning electron microscopy (JSM 6390,
161
Japan). Samples of micelles were dusted on an aluminum stub and coated with gold
162
using a cold sputter coater to a thickness of 400°A, and then imaged using a 20 Kv
163
electron beam.
164
2.5.4 Drug loading and entrapment efficiency
165
The amount of curcumin in the micelles was determined with UV-Vis spectrophotometer
166
(Model No. UV-1700, Shimadzu, Japan) at 423 nm. Prior to determination the micelles
167
were suitably diluted with alcohol. The percent drug-loading (DL %) and percent
Ac ce p
te
d
M
an
us
cr
ip t
147
8
Page 8 of 30
168
entrapment efficiency (EE %) of Curcumin in prepared micelles was calculated using the
169
following equations (Jansson, 2004).
170
% EE
171
amount of drug in micelles x100 total mass of drug loaded micelles
---- (3)
us
cr
ip t
=
_____ (4)
an
172
2.5.5 In vitro release study
174
The release of curcumin from the micelles was investigated by dialysis method. The in
175
vitro release of curcumin was achieved using PBS (pH 7.4) as the release medium.
176
µl of curcumin loaded micelles or curcumin solution (in propylene glycol) as control were
177
introduced in a dialysis membrane bag (Mol wt. cut-off 12000 Da). The bag was
178
suspended in 200 ml of phosphate buffer (pH 7.4) while being stirred maintained at 37 °C.
179
To assure the adequate sink condition; the whole release medium was replaced by a
180
fresh medium. The curcumin concentration in PBS was determined by UV
181
spectrophotometer, at wavelength of 423 nm (Ali Fattahi, 2012).
182
Drug release mechanism
183
In order to study drug release mechanism from curcumin loaded polymeric micelle, the
184
drug release data was fitted in Korsmeyer-Peppas equation. The logarithmic plot of
185
cumulative percentage of drug released vs. log time gives the release exponent n and K
186
value from the slope of the straight line and y intercept respectively. (Peppas, 1985).
200
Ac ce p
te
d
M
173
9
Page 9 of 30
187
Mt / M∞ = Ktn
188
2.6 In vitro assessment of developed dry powder inhaler
189
Freeze dried powder was subjected to Andersen cascade impactor (ACI, SS 316, Copley
190
Scientific Ltd.) studies to determine mass median aerodynamic diameter (MMAD) and
191
geometric standard deviation (GSD) (De Boer AH, et al. 2003; Jensen, et. al.2010; Muttil,
192
P, 2007). ACI utilizes eight jet stages make possible classification of aerosols and allows
193
airborne particulates to impact upon stainless steel impaction surfaces. Starting at the
194
filter, a cumulative mass deposition (under size in percentage) vs. cut-off diameter of
195
respective stages was derived, the mean mass aerodynamic diameter (MMAD),
196
geometric standard diameter (GSD), fine particle dose (FPD), % fine particle fraction
197
(%FPF) and % mass balance was calculated (Mahajan HS, Gundare SA, 2014).
198
2.7 In vivo studies:
199
All animal experiments were approved and performed in accordance with the guidelines
200
of by Institutional Animal Ethics Committee of R.C. Patel Institute of Pharmaceutical
201
Education and Research, Shirpur, resolution number RCPIPER/IAEC/2014-15/22. Male
202
Wistar rats of 6-8 weeks old weighing 250–270 g were selected for the in vivo analysis
203
studies which were divided into two group, one for intratracheal administration of the
204
formulation and another for intratracheal administration of the curcumin suspension.
205
a) Dose Administration:
206
The rats were anesthetized with an intraperitoneal injection of urethane (1 g/kg, 25% w/v)
207
and kept on a heating pad to maintain the body temperature. For intratracheal
208
administration anesthetized rats were placed on and trachea was exposed by blunt
Ac ce p
te
d
M
an
us
cr
ip t
------------------------------ (5)
10
Page 10 of 30
209
dissection sternohyoideus muscle and small midline incision was made over the trachea
210
between the fifth and sixth tracheal rings using a
211
cannulated with PEG 200 tubing (5-7cm) with the tip positioned approximately at the
212
bifurcation of trachea. Formulation (dose equivalent to 100mg/kg) were administered to
213
the Group 1 by inserting PEG tubing into, cannula which is attached with the micro
214
syringe with16 gauge needle. The powder was placed at the tip of needle and made 4-5
215
actuations of air pump to ensure the complete delivery of dose. Blood samples were
216
collected from tail vein intra-venously at 0.30, 1, 2, 4, 8, 16, 24, hrs. The rats were held at
217
45° position during intratracheal administration. To the Group 2 of curcumin suspension
218
(100mg/kg) was administered intratracheally by the same procedure mentioned above.
219
After administration the rats were sacrificed humanely at different time intervals and then
220
lungs were harvested by surgical resection. Both lung lobe was quickly rinsed with saline
221
and blotted up with filter paper to get rid of blood-taint and macroscopic blood vessels as
222
much as possible and weighed. Lungs were stored for up to 48 h in a deep freezer
223
(−70°C) until HPLC analysis (Misra et al., 2007, Beinborn et al., 2012).
224
b) Calibration curves of curcumin in lungs homogenate:
225
Calibration curves of curcumin were prepared with lung tissue homogenate by spiking the
226
known amounts of the drug utilizing its HPLC peak area ratio to the internal standard (4-
227
hydroxybenzophenone). The linear range of curcumin was 1–1000 ng/g for lung tissue.
228
The detection limits were 5ng/g for lung tissue samples.
229
C) Processing of samples:
230
i) Lung tissue:
Ac ce p
te
d
M
an
us
cr
ip t
20 guage needle followed by
11
Page 11 of 30
The lung tissue samples were homogenized with 1% w/w of saline in a tissue
232
homogenizer (Teflon homogenizer). To a 200 µlof lung homogenate, 50µl of the internal
233
standard (4-hydroxybenzophenone, 10 µg/mL) was spiked and vortex mixed for 30 s.
234
Then 0.5 ml of acetonitrile was added and vortex-mixed for 1 min. The sample was
235
centrifuged at 10000 rpm for 6 min at 4º C in ultracentrifuge (Beckman Coulter),
236
(Mukherjee et al., 2013). The supernatant layer (0.75 ml) was transferred to a 15 ml glass
237
test tube, and then 4.5 ml of extraction solvent, methyl t-butyl ether were added. The
238
sample was vortex-mixed for 3 min using a multi-tube vortex mixer and evaporated to
239
dryness under a stream of nitrogen at room temperature. 100 µl of supernant was
240
injected into the HPLC system after reconstitution.
241
ii) Chromatographic conditions:
242
The HPLC system (Agilent 1200 Series) consisted of C18 column (Eclipsed XDB 5 µm,
243
4.6 mm x 150 mm, Singapore), Ezchrome Elite Software, quaternary pump, Model G1354
244
A and Ultraviolet variable wavelength Diode Array detector, Model G1315D.
245
detection wavelength was 423 nm. The mobile phase was composed of acetonitrile–Citric
246
acid buffer (pH 3.0) (55:45) Elution was performed isocratically at 40°C at a flow rate of
247
1.0 mL/min (Zhimei Song, 2011).
248
iii) Pharmacokinetic analysis
249
The maximum plasma concentration (Cmax) and time of its occurrence (Tmax) were directly
250
computed from the plasma concentration v/s time plot. The Cmax, Tmax, and AUC were
251
computed using Kinetica 5® (Thermo Fisher Scientific) software. Relative bioavailability
252
(F) was calculated with reference to curcumin suspension.
The
Ac ce p
te
d
M
an
us
cr
ip t
231
12
Page 12 of 30
3.0 Results and discussion
254
3.1 Synthesis of grafted copolymer
255
Ring opening polymerization (ROP) reaction of L-lactide, xyloglucan and benzyl alcohol is
256
shown in Fig. 1. The graft copolymerization reaction has been carried out at in presence
257
a sacrificial initiator (benzyl alcohol), as the amount of initiating hydroxyl groups in
258
xyloglucan is uncertain. This co-initiator system has proved as an efficient method for ring
259
opening polymerization of lactides (Kricheldorf & Damrau, 1997; Lonnberg et al., 2006;
260
Trollsas et al., 2000). The time and temperature were kept constant during reaction.
261
Anionic ring-opening polymerization of cyclic ester monomers results of nucleophilic
262
attack of a negatively charged initiator on the carbonyl carbon or on the carbon atom
263
adjacent to the acyl-oxygen, resulting in linear polyester. The ROP reaction shows
264
formation of ester bond so it demonstrates that grafting of polymer was done successfully
265
(Andrew Maraisa et al, 2012).
266
3.2. Characterization of graft copolymer
267
3.2.1 IR spectra of xyloglucan and L-lactide grafted xyloglucan
268
The IR spectra of samples in KBr pellets were recorded using a FTIR spectrometer
269
(Shimadzu 8400S, Japan) in the range 450–4000 cm−1. FTIR spectra for xyloglucan and
270
grafted xyloglucan samples are shown in Fig. 2. The grafting of L-lactide chains from the
271
xyloglucan can evident in the FTIR spectra through the presence of new peak, around
272
1759 cm−1 in the carbonyl region.
273
3.2.2 NMR spectroscopy
Ac ce p
te
d
M
an
us
cr
ip t
253
13
Page 13 of 30
274
To confirm possible grafting of xyloglucan, 1H NMR spectra of xyloglucan and PLA
275
grafted xyloglucan was compared (Fig. 3). As illustrated in Fig. 3, the major chemical shift
276
of xyloglucan and grafted xyloglucan observed between 1 and 8 ppm. NMR spectra of
277
xyloglucan showed singlet at 2.1 (CH2) and multiplets at 3.91
278
OH), (O CH), 5.1 (O) and multiplets at 3.42, 3.76, due to CH (OH). NMR spectra of
279
modified polymer showed multiplets in the region 1.45 to 1.56
280
decreases intensity from 2.30 to 1.57 in the region of 3.57 to 3.74 proves successful
281
grafting of xyloglucan with poly lactic acid. (Silverstein et al, 2005).
282
3.2.3 In vitro Degradation
283
The weight remaining after degradation in-vitro was 62.85%. This phenomenon
284
demonstrated that the degradation and dissolution rate of grafted polymer was rapid SSF
285
in simulated body fluids under the normal body temperature.
286
3.2.4 In vivo degradation
ip t
(O CH2), 4.50 (O CH2
Ac ce p
te
d
M
an
us
cr
(OCO-CH3) and
287
After in vivo implantation in rats, the films showed weight loss of 90.23% with in 36
288
minutes. The bulk degradation is evident both in vitro and in vivo. Although the xyloglucan
289
and polylactic aciad are highly biodegradable polymer which presumably will lead to new
290
application of these polymers in the field of novel drug delivery system.
291
3.3 Determination of CMC and preparation of drug loaded polymeric micelles
292
As the structure of the grafted copolymers consists of hydrophobic and hydrophilic blocks,
293
hence the ability of micelle formation as reservoirs for drugs is of interest. The surface
294
tension of 0.1% solution polymer solution, measured by conventional stalagmometry
14
Page 14 of 30
method (number of drops of liquids between marks), has found to be 99.48 *10-3 N/m. To
296
calculate CMC from each curves two tangents were drawn to the point near the place
297
where the curves bended. The extrapolation of the crossing point of these tangents to the
298
abscissa axis gives the value of CMC. The critical micelle concentration of the grafted
299
xyloglucan micelle suspension was 1.5 X 10-2 % w/w.
300
3.4 Characterization of Curcumin loaded PLA-grafted xyloglucan micelles
301
3.4.1 Size distribution of polymeric micelles
us
cr
ip t
295
The mean particle size of the curcumin loaded micelles measured using the particle size
303
analyzer was 102.4 nm, with polydispersity index (PDI) of 0.275, signifying that the
304
narrow distribution of sizes of micelles. It is reported that the particle size will directly
305
influence carrier circulation time and distribution in vivo (Sezgin, et al. 2006). Small
306
particle sizes (<200 nm) could reduce the uptake of the reticulo endothelial system (RES)
307
and provide efficiently passive tumor-targeting ability via the enhanced permeability and
308
retention (EPR) effects (Dabholkar.et al 2006). Therefore, size of prepared curcumin
309
micelles was suitable for passive tumor targeting.
310
3.4.2 Zeta potential measurements
311
The zeta potential represents electrical charge to the micelle surface. It is important and
312
useful parameter to predict and control the stability of colloidal dispersion. The
313
measurement of zeta potential is an important tool to understand the dispersion
314
phenomenon. The zeta potential of pure drug, blank micelles and drug loaded micelles
315
were 9.69 mV, -21.7mV and -18.2 mV respectively. The greater the value, more likely the
316
colloidal system is to be stable because the charged particles repel one another and thus
Ac ce p
te
d
M
an
302
15
Page 15 of 30
overcome the natural tendency to aggregate. It is recognized that higher zeta potential
318
values, either positively or negatively charged, demonstrates that dispersion will have
319
greater long-term stability.
320
3.4.3 Morphological examination
321
Representative SEM image obtained for curcumin loaded polymeric micelles is shown in
322
Fig.4. The shape of dried micelles was mainly spherical and their size agreed well with
323
that measured by dynamic light scattering (DLS).
324
3.4.4 Drug loading and entrapment efficiency
325
Entrapment of hydrophobic drug into micelles was results of chemical conjugation or
326
physical entrapment. During physical entrapment of drug in micelles various forces
327
involved including hydrogen bond and van-der Waals forces. Curcumin was encapsulated
328
into Xyloglucan grafted L-lactide micelles with loading capacity of 68.9 ± 0.02% and
329
entrapment efficiency of 96.38± 0.2% respectively. The lower drug loading was attributed
330
to limited aqueous solubility of grafted copolymer.
331
3.4.5 In vitro drug release study
332
In vitro release of curcumin from micelle formulation under sink condition was investigated
333
by dialysis method PBS (pH 7.4) release medium. As shown in Fig. 5A, at the end of 5 h
334
about 50% of curcumin released from polymeric micelles, where as about 75% of
335
curcumin was released from the propylene glycol solution. The results showed that
336
curcumin follows a sustained-release property from polymeric micelle that was similar to
337
previously reported studies. The slope of the plot (Fig 5B) was 0.81 demonstrating the
Ac ce p
te
d
M
an
us
cr
ip t
317
16
Page 16 of 30
anomalous (non fickian) release kinetic i.e. release of curcumin from micelles followed
339
erosion- diffusion mechanism (Peppas NA, Shalin JJ, et al 1989).
340
In vitro assessment of dry powder inhaler
341
The freeze dried powder micelles were subjected for the in vitro performance and
342
aerodynamic property using Anderson cascade impactor. The data obtained for various
343
aerodynamic properties demonstrates that 0.4% drug retained in the capsule and device
344
that has given indication of good dispersibility of formulated DPI and about 99.6% of drug
345
emitted from DPI. Fine particle fraction (FPF) was found to be 12.16%. The Mass Median
346
Aerodynamic Diameter (MMAD) of the curcumin loaded micelles was 106.67nm and was
347
less than 5μm which is prerequisite to have an inhalation of powders into the lower region
348
of the lung. The geometric standard diameter (GSD) found to be 68.77 nm, which was
349
depending on aerodynamic behavior.
350
In vivo studies
351
The in vivo studies conducted in this investigation to test the possibility of assessing
352
targeting ability of novel grafted polymer micelles to lungs. The drug distribution
353
parameters in lungs were calculated and are represented Table 1. The Cmax was
354
50342±1088.8 ng/ml at Tmax of 180 min for curcumin suspension and 89301±8182.1
355
ng/ml at Tmax of 120 min for powder micelle formulation respectively. Eventually, there
356
was an increase in AUC0-480
357
suspension thereby confirms the maintenance of effective drug concentration in lung
358
tissue for prolonged period as compared to suspension due to the ability of micelles in
359
reaching the pulmonary region. The relative bioavailability of curcumin when administered
360
as micelles formulation was about 539 ± 9.86, reflecting the potential use of micelles as
Ac ce p
te
d
M
an
us
cr
ip t
338
min
for DPIs compared with the AUC0-480min of curcumin
17
Page 17 of 30
novel carrier for the poorly bioavailable curcumin.
362
4.0 Conclusion
363
Newly synthesized L-lactide grafted xyloglucan copolymer can self-assemble to form
364
micelles with 0.0150 wt% CMC. Average particle size was 102.4 nm with 0.275 PDI. The
365
polymeric micelles, exhibited higher entrapment efficiency and drug loading for curcumin.
366
In vitro dry powder for inhalation performance observed for developed freeze dried
367
micelles suggesting high deep lung deposition of drug. The data of in vivo studies showed
368
high and prolonged residence of curcumin within lungs after pulmonary administration.
369
Novel grafted micelles expected to provide improved local concentration. The results of
370
present study clearly indicated promising potential of novel grafted polymer micelles for
371
delivering curcumin to deep lungs for effective treatment of lungs cancer.
372
Acknowledgement
373
Contributors are grateful to DSP Gokyo Food and Chemical Ltd. (Fukusima, Japan) for
374
gifting xyloglucan and Dr.S.J.Surana (Principal R.C.Patel IPER Shirpur) for providing
375
essential facilitates required carrying out research work.
376
5.0 References
Ac ce p
te
d
M
an
us
cr
ip t
361
377
Ali Fattahia, Mohammad-Ali Golozara, Jaleh Varshosazb, Hamid Mirmohammad
378
Sadeghid, Mohammadhossein Fathia. (2012).Preparation and characterization of
379
micelles of oligomeric chitosan linked to all-trans retinoic acid, Carbohydrate
380
Polymers. 87, 1176– 1184.
18
Page 18 of 30
Andrew Maraisa, Joby J. Kochumalayil., (2012). Toward an alternative compatibiliser
382
for PLA cellulose composites: Grafting Of xyloglucan with PLA, Carbohydrates
383
polymer, 89, 1038-1043.
384
Auras, R., Harte, B., & Selke, S. (2004). An overview of polylactides as packaging
385
materials. Macromolecular Bioscience, 4(9), 835–864.
386
Avachat, AM., Gujar, KN., Wagh, KV. (2013).Development and evaluation of tamarind
387
seed
388
Carbohydrate Polymers, 91, 537–542.
389
Bahl AB, Bahl BS, Tuli GD.(2011) Essentials of Physical Chemistry ,Multicolor Edition
390
S Chand Publishers.428-429..
391
Beinborn, NA, Wiederhold, NP, Hugh, DC and Williams (2012). Dry powder
392
insufflation of crystalline and amorphous voriconazole formulations produced by thin
393
film freezing to mice. European Journal of Pharmaceutics and Biopharmaceutics, 81,
394
1, 600–608.
395
Buckeridge, MS., Santos, HP, Tine, M.A.S. (2003). Mobilization of storage cell wall
396
polysaccharides in seeds. Plant Physiology and Biochemistry, 8(1–2), 141–156.
397
Carpita, NC. , Gibeaut, DM. (1993). Structural models of primary cell walls in flowering
398
plants: Consistency of molecular structure with the physical properties of the walls
399
during growth. Plant Journal, 3(1), 1–30.
400
Dabholkar, RD., Sawant, RM., Mongayt, DA, Devarajan, PV, Torchilin, VP. (2006).
401
Polyethylene glycol-phosphatidyl ethanolamine conjugate (PEG-PE)-based mixed
buccal
films
of
rizatriptan
benzoate.
M
an
mucoadhesive
Ac ce p
te
d
xyloglucan-based
us
cr
ip t
381
19
Page 19 of 30
micelles: some properties, loading with paclitaxel, and modulation of P-glycoprotein-
403
mediated efflux, International Journal of Pharmaceutics. 315, 148–157.
404
Gogolewski, S, Jovanovic, M, Perren, SM, Dillon, JG, Hughes, MK. (1993). Tissue
405
response and in vivo degradation of selected polyhydroxyacids: polylactides (PLA),
406
poly (3-hydroxybutyrate) (PHB) and poly (3-hydroxybutyrate-cp-3-hydroxyvalerate)
407
(PHB/VA).Journal of Biomedical Material Research.27, 1135-1148.
408
Jansson, J. Schille, K. Olofsson, G. Cardoso, R. Loh, W. (2004). The interaction
409
between PEO–PPO–PEO triblock copolymers and ionic surfactants in aqueous
410
solution studied using light scattering and calorimetry, Journal of Physical Chemistry.
411
B 108, 82–92.
412
Jiang, M., Wu, Y., He, Y., Nie, J. (2009). Micelles formed by self-assembly of hyper-
413
branched poly [(amine-ester)-co-(d,l-lactide)] (HPAE-co-PLA) copolymers for protein
414
drug delivery. Polymer International, 58(1), 31–39.
415
Kohori, F., Sakai, K., Aoyagi, T., Yokoyama, M., Sakurai, Y., Okano, T.
416
(1998).Preparation and characterization of thermally responsive block copolymer
417
micelles comprising poly (N-isopropyl acrylamide-b-dl-lactide). Journal of Controlled
418
Release, 55(1), 87–98.
419
Kricheldorf, HR., & Damrau, DO. (1997). Polylactones. 37. Polymerizations of l-lactide
420
initiated with Zn (II) l-lactate and other resorbable Zn salts. Macromolecular Chemistry
421
and Physics, 198(6), 1753–1766.
422
Kumar R., Srivastava A., Behari, K. (2009). Synthesis and characterization of
423
polysaccharide
Ac ce p
te
d
M
an
us
cr
ip t
402
based
graft
copolymer
by
using
potassium
peroxymonosul-
20
Page 20 of 30
phate/ascorbic acid as an efficient redox initiator in inert atmosphere. Journal of
425
Applied Polymer Science, 112(3), 1407–1415.
426
Kwon, GS. Forrest, ML. (2006). Amphiphilic block copolymer micelles for nanoscale
427
drug delivery. Drug Development Research, 67(1), 15–22.
428
Letchford, K., Burt, H. (2007). A review of the formation and classification of
429
amphiphilic block copolymer nanoparticulate structures: Micelles, nanospheres,
430
nanocapsules and polymersomes. European Journal of Pharmaceutics and
431
Biopharmaceutics, 65(3), 259–269.
432
Lonnberg, H., Zhou, Q., Brumer, H., Teeri, T.T., Malmstrom, E., Hult, A. (2006).
433
Grafting of cellulose fibers with poly (epsilon-caprolactone) and poly(l-lactic acid) via
434
ring-opening polymerization. Biomacromolecules, 7(7), 2178–2185.
435
Maeda, T., Yamashita, H., Morita, N. (2007). Application of xyloglucan to improve the
436
gluten membrane on bread making. Carbohydrate Polymers, 68, 658–664.
437
Mahajan HS. Gundare SA. (2014). Preparation, characterization and pulmonary
438
pharmacokinetics of xyloglucan microspheres as dry powder inhalation. Carbohydrate
439
Polymers.102, 529–536.
440
Mishra, MM. Tripathy, J, Yadav, M., Sand, A., & Behari, K. (2010). Graft copolymer (K-
441
carrageenan-g-N-vinyl formamide): Preparation, characterization and application.
442
Carbohydrate Polymers, 80, 235–241,
Ac ce p
te
d
M
an
us
cr
ip t
424
21
Page 21 of 30
443
Misra, A, Chougule, M, Padhi, B. (2007). Nano-liposomal dry powder inhaler of
444
tacrolimus:
445
International Journal of Nanomedicine, 2, 4, 675–688.
446
Mukherjee, B, Sinha, B, Pattnaik, G (2013). Poly-lactide-co-glycolide nanoparticles
447
containing voriconazole for pulmonary delivery: in vitro and in vivo study, Nanomed:
448
Nanotech, Bio, Med, 9, 1, 94–104.
449
Peppas NA, Shalin, J.J. (1989). A simple equation for the description of solute release
450
III. Coupling of diffusion and relaxation. International Journal of Pharmaceutics, 57,
451
169-172.
452
Peppas, NA (1985), Analysis of fickian and non fickian drug release from polymers’,
453
Pharm ActaHelv, vol. 60, 110-111.
454
Rao, PS., Srivastava, HC. (1973). Industrial gums: Polysaccharides and their
455
derivatives. Diego: Academic Press. 369–411.
456
Satturwar, PM, Fulzele, SV, Dorle, AK. (2003) Biodegradation and In vivo
457
biocompatibility of rosin: a natural film-forming polymer. AAPS PharmSciTech 4, 1-7.
458
Sezgin, Z.
459
polymeric micelles for solubilization of poorly soluble anticancer drugs, European
460
Journal of Pharmaceutics and Biopharmaceutics. 64, 261–268.
461
Shi, R, Zhu, A, Chen, D., (2010) In vitro degradation of starch/PVA films and
462
biocompatibility evaluation. Journal of Applied Polymer Science, 115, 346-357.
characterization,
and
pulmonary
Pharmacokinetics,
Ac ce p
te
d
M
an
us
cr
ip t
Preparation,
Yuksel, N. Baykara, T. (2006). Preparation and characterization of
22
Page 22 of 30
Silverstein, RM., Webster, FX., Kiemle, DJ. (2005). Spectrophotometric identification
464
of organic compounds. John Wiley and Sons, 150–151.
465
Suggs, LJ, Krishnan, RS, Garcia, CA, Peter, SJ, Anderson, JM, Mikos, AG. (1998) In
466
vitro and in vivo degradation of poly(propylene fumarate co-ethylene glycol) hydrogels.
467
Journal Biomedical material Research, 42, 312-320.
468
Tripathy, J., Mishra, D. K., Yadav, M., Sand, A., Behari, K. (2009). Modification of -
469
carrageenan by graft copolymerization of methacrylic acid: Synthesis and applications.
470
Journal of Applied Polymer Science, 114(3), 3896–3905.
471
Trollsas, M., Claesson, H., Atthoff, B., Hedrick, JL, Pople, JA, Gast, AP. (2000).
472
Conformational and structural properties of high functionality dendrimer-like star
473
polymers
474
Symposia, 153, 87–108.
475
Tsuji, H. (2005). Poly (lactide) stereo complexes: Formation, structure, properties,
476
degradation, and applications. Macromolecular Bioscience, 5(7), 569–597.
477
Zhimei Song, (2011). Curcumin-loaded PLGA-PEG-PLGA triblock copolymeric
478
micelles: Preparation, pharmacokinetics and distribution in vivo, Journal of Colloid and
479
Interface Science, vol. 354, 116–123.
living polymerization techniques. Macromolecular
d
from
Ac ce p
te
synthesized
M
an
us
cr
ip t
463
480 481
Figure legends
482
Fig 1: Scheme of grafting of xyloglucan with L-lactide by Ring Opening Polymerization
483
(ROP)
484
Fig 2: FTIR spectra of native (A) and grafted xyloglucan (B) 23
Page 23 of 30
485
Fig 3: NMR spectra of xyloglucan (bottom) and grafted xyloglucan (top)
486
Fig 4: Scanning electron microscopic images of polymeric micelles
487
Fig 5: In vitro drug release from polymeric micelles (A), drug release mechanism (B).
ip t
488 489
Table 1: Pharmacokinetic parameters following intratracheal administration curcumin
491
micelles as dry powder inhaler formulation and suspension. Suspension 50342 ± 1088.8
an
Cmax (ng/ml) Tmax (min)
89301± 8182.1 120
24132010±90183.4
53241082±403215.0
--
539 ± 9.86
492 493
Ac ce p
HIGHLIGHTS
te
d
Relative bioavailability
Micelles as DPI
180
M
AUC 0-480 min (ng/ml*min)
494
us
Pharmacokinetic Parameter
cr
490
495
PLA grafted xyloglucan copolymer was synthesized successfully by ring opening
496
polymerization.
497
IR and NMR analysis results reveal successful grafting of PLA with xyloglucan.
498
Produced micelle showed suitable particles size, CMC, drug loading and release
499
efficiency.
500
PLA grafted xyloglucan based micelles are promising drug carriers.
501 502 24
Page 24 of 30
503 504
te
d
M
an
us
cr
ip t
Ac ce p
505
25
Page 25 of 30
Ac
ce
pt
ed
M
an
us
cr
i
Figure(s)
Page 26 of 30
Ac
ce
pt
ed
M
an
us
cr
i
Figure(s)
Page 27 of 30
Ac ce p
te
d
M
an
us
cr
ip t
Figure 3
Page 28 of 30
Ac
ce
pt
ed
M
an
us
cr
i
Figure 4
Page 29 of 30
Ac
ce
pt
ed
M
an
us
cr
i
Figure(s)
Page 30 of 30