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Title: CHITOSAN-STARCH NANOCOMPOSITEPARTICLES AS A DRUG CARRIER FOR THE DELIVERYOF BIS-DESMETHOXY CURCUMIN ANALOGUE
Author: Sindhuja Bala Subramanian Arul Prakash FrancisThiyagarajan Devasena
PII: S0144-8617(14)00737-1DOI: http://dx.doi.org/doi:10.1016/j.carbpol.2014.07.053Reference: CARP 9109
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Received date: 11-3-2014Revised date: 15-7-2014Accepted date: 17-7-2014
Please cite this article as: Subramanian, S. B.,CHITOSAN-STARCHNANOCOMPOSITE PARTICLES AS A DRUG CARRIER FOR THE DELIVERYOF BIS-DESMETHOXY CURCUMIN ANALOGUE, Carbohydrate Polymers(2014), http://dx.doi.org/10.1016/j.carbpol.2014.07.053
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CHITOSAN-STARCH NANOCOMPOSITE PARTICLES AS A DRUG CARRIER 1
FOR THE DELIVERY OF BIS-DESMETHOXY CURCUMIN ANALOGUE2
Sindhuja Bala Subramaniana,b, Arul Prakash Francisa,c, Thiyagarajan Devasenaa,d3
4
aCentre for Nanoscience and Technology, A.C. Tech Campus, Anna University, 5Chennai- 600025, India6
dCorresponding author:9
Thiyagarajan Devasena10
Centre for Nanoscience and Technology11
A.C.Tech Campus12
Anna University 13
Chennai – 60002514
India.15
Email: [email protected]
Mobile: +91 996264515117
Fax: +91 44 2230165618
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ABSTRACT24
The conventional drug delivery system has serious limitations such as lack of target 25
specificity, altered effects and diminished potency. These limitations can be 26
overcome by using biocompatible polymer as an effective drug delivery system. In 27
this study, bis-demethoxy curcumin analogue loaded Chitosan-starch (BDMCA-CS) 28
nanocomposite particles were developed using different ratios of Chitosan and starch 29
(3:1, 1:1 & 1:3) by ionic gelation method. The entrapment efficiency and drug loading 30
capacity were found to be high for the formulation with the ratio 3:1 of BDMCA: CS. 31
Physical characterization of the nanocomposite particles was determined using DLS 32
and FTIR. The morphology of the BDMCA-CS nanocomposite particles were found to 33
be spherical and regular by SEM analysis. In-vitro drug release profile of the 34
BDMCA-CS nanocomposite particles showed a very slow and sustained diffusion 35
controlled release of the drug. The cancer cells targeting ability of the BDMCA-CS 36
nanocomposite particles were confirmed by performing MTT assay on MCF-7 Breast 37
cancer cell lines and VERO cell lines.38
Keywords: Drug delivery, nanocomposite, Chitosan, starch, BDMCA, MTT assay39
1. INTRODUCTION40
Nanoparticles are usually referred as particles with a size ranging from 1 to 100nm. 41
They have completely new properties based on size, distribution and morphology42
(Jhan, 1999). Nanotechnology offers biological and clinical applications in terms of 43
diagnosis, controlled drug delivery and imaging. It is possible to accumulate them for 44
an extended period of time inside a cell which can improve diagnostic sensitivity and 45
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therapeutic efficiency (Vidyanathan et al., 2009). These particles can penetrate 46
across many biological barriers through small capillaries into individual cells, allowing 47
a drug to accumulate at a target site in the body. But in addition to the biological 48
applications, use of metal oxide nanoparticles in drug delivery could lead to oxidative 49
stress, inflammation and damage to blood-brain barrier (BBB) (Francis et al., 2011) in 50
contrast to biocompatible and biodegradable non-toxic polymer. Therefore, there is a 51
need for biocompatible nanoparticles with higher beneficiary effects and lower toxic 52
nature.53
Polymer nanoparticles offer the best results by encapsulating drugs and shows 54
reduced toxicity by limiting the interaction of with healthy cells. Such delivery systems55
also have potential benefits such as controlled and long term release rates, 56
enhanced bioactivity, reduced side effects, decreased administration frequency and 57
ability to deliver multiple drugs to the same site. For example, chitosan nanoparticles 58
have been reported as drug carrier for 5-Fluorouracil (Nagarwal et. al, 2011) and 59
isoniazid (Rafeeq et al., 2010). Chitosan, the partially acetylated mucoadhesive and 60
cationic (1-4)-2-amino-2-deoxy-β-D-glucan, is being used extensively as a drug 61
carrier owing to its biocompatibility and biodegradability (Busilacchi et al., 2013; 62
Muzzarelli, 2009; Muzzarelli, 2010; Muzzarelli et al., 2012; Thanou, et al., 2001). It 63
has antimicrobial and antitumor activities (Qin, et al., 2002; Muzzarelli et al., 1990; 64
Roller & Covill, 1999; Zheng & Zhu, 2003), owing to its unique characteristics, such 65
as i) higher affinity for negatively charged biological membranes and ii) site-specific 66
targeting in vivo, respectively (Calvo, et al., 1997; Qi, et al., 2004). By maintaining the 67
optimum size, it accumulates in the tumor tissue via specific surface recognition 68
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(Debnath, et al., 2010). Previous studies have also reported that a combination of 69
alginate, chitosan and pluronic F127 loaded with curcumin have improved properties 70
such as solubility, encapsulation and dispersity of the drug (Das et al., 2010) and 71
chitosan -dextran sulphate complex have shown enhanced stability and increased 72
mechanical strength (Chen et al., 2007) than when used as individual polymer. 73
Starch is a natural storage polymer of plants with unique properties such as 74
biodegradability, low cost and renewability. It has been recently reported that starch 75
based polymers have greater application in the biomedical field such as drug delivery 76
systems and tissue engineering scaffolds (Baran et al., 2004). The major drawback of 77
starch is that it is poor in processability, dimensional stability and mechanical 78
properties of its end products. Thus, starch has been reported to be used as a 79
nanocomposite (Lu et al., 2009). It was interesting to note that the mechanical 80
strength of chitosan-TPP beads increases on addition of starch granules (Venugopal, 81
2011). Bajer et al have reported the intermolecular interactions between the chitosan 82
and starch in their blend (Bajer & Kaczmarek, 2010). Thus we expect the formation 83
of chitosan-starch composite carriers which may load the anticancer drug (BDMCA) 84
and subsequently release the drug with high efficacy. 85
BDMCA stands for Bis-desmethoxycurcumin analogue, which is a curcuminoid of 86
turmeric. In vitro and animal studies have reported that curcuminoids has antitumor87
(Aggarwal & Shishodia, 2006; Choi, et al., 2006; Strofer, et al., 2011), antioxidant, 88
antiarthritic, antiamyloid, anti-ischemic (Shukla, et al., 2008) and anti-inflammatory 89
properties (Stix, 2007). It has potential anticancer activity which stems from its ability 90
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to induce apoptosis in cancer cells without cytotoxic effects on healthy cells91
(Aggarwal & Shishodia, 2004). Studies have reported that Curcumin analogs show 92
poor bioavailability due to poor absorption, rapid metabolism and rapid systemic 93
elimination (Anand, et al., 2007). Therefore, there is a need for controlling release of 94
this drug, through which good bioavailability can be achieved rapidly even at a lower 95
dose.96
Therefore, this investigation aims to synthesize a polymer nanocomposite using 97
chitosan and starch loaded with BDMCA by ionic gelation technique and to determine 98
(i) the drug (BDMCA) loading efficiency of the CS nanocomposite (ii) the drug 99
entrapment efficiency of the CS nanocomposite (iii) the anticancer activity of 100
BDMCA-CS nanocomposite against MCF-7 breast cancer cell line by MTT assay and 101
(iv) cytotoxic activity of BDMCA-CS nanocomposite against VERO cell lines by MTT 102
assay.103
2. MATERIALS AND METHODS104
2.1 Materials105
Chitosan, starch and sodium tri poly phosphate were purchased from Sigma-Aldrich. 106
Soluble starch was obtained from Hi Media and Tween-80 from Merck Specialties 107
Pvt. Ltd., Mumbai and Glacial acetic acid from Thermo Fischer Scientific India Pvt. 108
Ltd. Acetyl acetone, Dimethyl formamide, Diethanolamine and boric acid were 109
purchased from Sisco Research Laboratories Pvt. Ltd. Salicylaldehyde was obtained 110
from Merck Specialities Pvt. Ltd. 111
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2.2 Methods112
2.2.1 Preparation of BDMCA-CS nanocomposite particles113
The chitosan-starch (CS) nanocomposite particles were synthesized using ionic 114
gelation method (Calvo et al., 1997). Chitosan and soluble starch solutions were 115
prepared in 2% Acetic acid. After the complete dissolution of chitosan and starch in 116
acetic acid, few drops of 0.5% Tween-80 were added to the solution for uniform 117
dispersion and to prevent aggregation at ambient temperature during stirring. 118
BDMCA (Bis-desmethoxy Curcumin Analogue) was prepared using the protocol 119
reported earlier (Babu & Rajasekaran, 2009). Briefly, to the mixture of acetyl acetone 120
and boric acid, dimethyl formamide (DMF) was added in drops and heated for 15 121
minutes. This mixture was heated with salicylaldehyde for 5 minutes. A few drops of 122
glacial acetic acid and diethanolamine mixture was added to this mixture and heated 123
for 5 to 6 hrs. Kept it for overnight and then DMF was added and warmed to form a 124
flow paste. This paste was added slowly into the 10% acetic acid and stirred for 2 to 125
3 hrs. After stirring, the mixture was sonicated for 15 mins. Then the mixture was 126
filtered in a Whatman No 1 filter paper using a vacuum pump and dried on a watch 127
glass at room temperature for 1-2 hours. About 0.05% of chemically synthesized 128
BDMCA was added to the CS nanocomposite. Finally, 1% sodium tri poly phosphate 129
was added in drops to the solution as a cross linking agent until opalescence. The 130
final suspension was centrifuged. The pelleted particles were then lyophilized at 5 131
mTorr and -54�C for 8 hours. These lyophilized particles were then denoted as 132
BDMCA-CS nanocomposite particles. The BDMCA-CS nanocomposite particles were 133
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characterized by Particle size analysis, Scanning electron microscopy, Fourier 134
transform infra-red spectroscopy. The BDMCA-CS nanocomposite particles were 135
also analyzed for drug loading efficiency, in vitro drug release studies, cytotoxic 136
activity and anticancer activity. The supernatant was used for the determination of 137
drug entrapment efficiency. Different formulations of BDMCA-CS nanocomposite 138
particles were prepared as shown in Table.1.139
Table.1 Different formulations of BDMCA loaded Chitosan-Starch (CS) 140
nanocomposite particles141
Formulation Code Chitosan
(mg)
Starch
(mg)
STPP
(%)
Tween-80
(%)
BDMCA
(mg)
BDMCA-CS 3:1 150 50 1 0.5 50
BDMCA-CS 1:1 100 100 1 0.5 50
BDMCA-CS 1:3 50 150 1 0.5 50
142
2.2.2 Physical Characterization143
The lyophilized BDMCA-CS nanocomposite particles were dispersed in distilled water 144
and its particle size was measured using MALVERN ZETASIZER.145
The scanning electron microscope (HITACHI model S3400N, Japan) was used to 146
determine the surface morphology of nanocomposite particles. A drop of the well-147
dispersed sample was placed on a disc which was then air dried and scanned in a 148
high vacuum chamber with a focused electron beam. 149
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The infrared absorption bands of chitosan, starch, CS, BDMCA and BDMCA-CS 150
nanocomposite particles were obtained using PERKIN ELMER Spectrum Rx1, USA. . 151
1mg each of chitosan, starch, CS, BDMCA and BDMCA-CS nanocomposite were152
ground in a mortar pestle along with KBr to make a small pellet of thickness 0.1cm. 153
KBr pellet was used as blank. Spectrum was observed in the scanning range of 400 to 154
4,500 cm-1 with a resolution better than 0.7 cm−1 resolution. The peaks were used to 155
identify the molecular components and structures. 156
2.2.3 Percentage drug content and drug encapsulation efficiency157
About 2mg of the lyophilized BDMCA-CS nanocomposite particles were dissolved in 158
10ml Acetonitrile and centrifuged at 8000rpm for 10 minutes. The acetonitrile 159
solubilize the BDMCA alone to bring them out of the nanocapsules to be released 160
into the supernatant. The absorbance of the supernatant was analyzed using UV-161
Visible Spectrophotometer (T90+ UV/VISIBLE Spectrometer PG Instrument Ltd.) at 162
418nm. The amount of BDMCA present in the supernatant was calculated using the 163
calibration curve. From the calculated concentration of BDMCA, the percentage drug 164
content was determined for all the three different formulations of BDMCA-CS using 165
the following formula:166
167
168
169
170
171
Total amount of drug × 100
Percentage drug content =
Total weight of BDMCA-CS particles
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After the incorporation of 0.05% BDMCA into the nanocomposite particles, the 172
solution was centrifuged at 8000rpm for 10 minutes. The photometric absorbance of 173
the supernatant containing unloaded drug molecules was determined at the 174
wavelength of 418 nm. The amount of BDMCA present in the supernatant was 175
calculated by the formula: Y= 0.0084X + 0.011 using the calibration curve. From the 176
calculated concentration of BDMCA, the encapsulation efficiency of the 177
nanocomposite particles was determined for the three different formulations of 178
BDMCA-CS nanocomposite particles using the following formula:179
180
181
The best two formulations of BDMCA-CS nanocomposite particles showing the high 182
percentage drug contentand high drug encapsulation efficiency were taken for in vitro 183
drug release studies.184
185
2.2.4 In vitro drug release studies186
In vitro drug release studies of the BDMCA-CS nanocomposite particles were 187
performed in a Franz-Diffusion cell containing phosphate buffer (pH 7.4) and using a 188
dialysis membrane with a molecular weight cutoff (MWCO) of 10 kDa. The 189
membrane was placed over the mouth of the cell, to which the nanocomposite 190
particles were loaded. A magnetic peddle was placed in the cell and maintained at 191
100 rpm in a magnetic stirrer for uniform mixing. The entire setup was placed in a 192
(Total amount of drug – Amount of unbound drug) × 100
Encapsulation efficiency =
Total amount of drug
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bowl filled with distilled water to maintain the temperature at 37.4ºC. About 2ml of the 193
solution was removed, using a syringe, from the Franz-Diffusion cell for every one 194
hour and replaced with 2ml of fresh phosphate buffer. The absorbance of the 195
collected solution was measured using UV-Visible Spectrophotometer at 418nm. 196
From the absorbance value, the concentration of the BDMCA in the solution using a 197
standard calibration curve. The formulation which shows slow and sustained drug 198
release profile was taken for cytotoxic and anticancer assay studies.199
2.2.5 Drug release kinetics200
In order to understand the mechanism and kinetics of drug release, it is essential to 201
fit the results of in vitro drug release study of well-known kinetic equations such as 202
Zero order model (%Cumulative drug release Vs Time), First order model (Log % 203
cumulative drug remaining Vs Time), Higuchi model (% cumulative drug release Vs 204
square root of time) and Hixson-Crowell model (Cube root of % cumulative drug 205
remaining Vs Time). Since our formulation is a polymeric system, the drug release 206
data were further analyzed using Korsmeyer-Peppas model equation, Mt/M∞ = Ktn, 207
where Mt/M∞ is the fraction of drug released at time t, k is release rate constant and n 208
is the release exponent (Dash, et al., 2010). 209
2.2.6 Anticancer assay 210
The anticancer activity of the BDMCA-CS nanocomposite particles were checked on 211
MCF-7 Breast cancer cell lines and on VERO cell lines (kidney epithelial cells from 212
African green monkey). Cells (1 × 105/well) were plated in 100 μl of medium (MEM) 213
per well in 96 well titre plates (Hi media). After 48 hours incubation at 37ºC the cell 214
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reaches the confluence. Then, the medium was removed and the monolayer of cells 215
was washed twice with MEM. To the washed cell sheet 1ml of the medium containing 216
defined concentrations of BDMCA-CS nanocomposite particles were added in 217
respective wells. Each dilutions of BDMCA-CS nanocomposite ranged from 1:1 to 218
1:64 and they were added to the respected wells of 96 well titre plates. The plates 219
were incubated for 48h at 37°C in 5% CO2. After incubation, the wells were decanted 220
and washed with MEM for 2 to 3 times. Then, about 5mg/ml concentration of MTT (3-221
(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl--tetrazolium bromide) was added into the well 222
(200µl/well). After 4h incubation, 100µl of DMSO was added to each well and was 223
incubated for 10 minutes. The presence of viable cells was then confirmed by the 224
formation of purple color formazan crystals after adding solubilizing reagent (DMSO). 225
The absorbance was measured at 594 nm colorimetrically. Cell viability was defined 226
as the ratio (expressed as a percentage) of absorbance of treated cells to untreated 227
cells. 228
% Cell Viability = (Test Absorbance / Control Absorbance) × 100229
The relative % Cell Viability was obtained by plotting concentration along X- axis and 230
cell viability along Y- axis.231
232
233
234
235
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3. RESULTS 236
3.1 Particle size analysis (PSA)237
The particle sizes of the BDMCA-CS nanocomposite particles of different 238
formulations (3:1, 1:1 and 1:3) are shown in Table.2 and their dispersity is shown in 239
Figure.1a, 1b &1c respectively.240
Table.2 Size distribution of different formulations of BDMCA-CS 241
nanocomposite particles242
243
244
245
246
A maximum size of 320 nm was observed in the formulation BDMCA-CS 3:1 due to 247
the higher concentration of Chitosan. A size of 170 nm was observed in BDMCA-CS 248
1:3 due to increased concentration of Starch. Uniform size distribution was obtained 249
in all the three formulations. However, as the chitosan and starch concentration was 250
reduced, the size of the nanocomposite particles got reduced with the least size of 251
91nm in the formulation BDMCA-CS 1:1. 252
Formulation code Size of the particle (nm)
BDMCA-CS 3:1 320
BDMCA-CS 1:1 91
BDMCA-CS 1:3 170
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253
Figure 1. Size dispersity of BDMCA-CS nanocomposite particles of ratio (a) 3:1, (b) 254
1:1 & (c) 1:3255
3.2 Scanning electron microscopy (SEM)256
The SEM images of all the three formulations (BDMCA-CS 3:1, 1:1 & 1:3) are shown 257
in Figure.2a, 2b & 2c. The morphology of the BDMCA-CS nanocomposite particles 258
were found to be spherical with smooth and regular surface for all the three 259
formulations. The change in polymer concentration does not affect the morphology. 260
Amongst all shapes spherical nanoparticles are best suited for drug delivery 261
applications. (Venkatraman, James, Zhan & Yang 2011). Reports suggest that the 262
spherical nanoparticles have higher probability for cell entry (Zhang, X. Q., Xua, X., 263
Bertrand, N., Pridgenb, E., Swamia, A., & Farokhzad O.C. 2012). Therefore, we 264
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report that the three formulations may be investigated for drug loading and drug 265
encapsulation efficiency.266
267
Figure 2. SEM image of different ratios of nanocomposites (a) BDMCA-CS 3:1 (b) 268
BDMCA-CS 1:1 (c) BDMCA-CS 1:3269
3.3 Fourier transform infrared spectroscopy (FTIR)270
The IR spectrums of chitosan, starch, CS, BDMCA and BDMCA-CS nanocomposite 271
particles were reported in the Figure.3272
The IR spectrum of chitosan showed five characteristic peaks at 3451cm-1 (-OH 273
stretching), 2920 cm-1 (-CH stretching), 1647cm-1 (-NH2), 1382 cm-1 (-CO stretching 274
of primary alcoholic group) and 1076cm-1 (C-O-C stretching). These results coincide 275
with the findings of Nagarwal et al. (2011).276
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The spectrum of starch showed peaks at 3402cm-1 (-OH stretching), 2929cm-1 (-CH 277
stretching), 1159cm-1 (-CO stretching), 997cm-1 and 925cm-1 (-COH bending), 278
1084cm-1 and 859cm-1(C-O-C symmetrical stretching). These results are found to be 279
similar to the data reported earlier (Cael, Koenig & Blackwell, 1973; Cael, Koenig & 280
Blackwell, 1975).281
On comparison of IR spectrum of chitosan and starch with that of CS nanocomposite 282
particles, we noticed that the absorption band owing to OH moiety has broadened at 283
3482cm-1. Kumari et al. (2011) has implied that broadening of the OH stretch is an 284
indication of intermolecular hydrogen bonding. We could, therefore, suggest that the 285
chitosan is hydrogen bonded to starch. The 1647cm-1 peak of –NH2 group in chitosan 286
shifts to 1654cm-1.This indicates that the phosphate group of tripolyphosphate is 287
associated with ammonium groups of chitosan through electrostatic interactions to 288
form the polyelectrolyte complex. Overall, the FTIR data of chitosan, starch and CS 289
nanocomposite confirms that the spherical particles (observed in SEM) are 290
nanocomposite of chitosan and starch, gelated with TPP.291
The FTIR spectrum of BDMCA shows O-H stretching at 3425cm-1, C=O stretching at 292
1614cm-1and 1230cm-1, C=C olefienic stretching and C=C aromatic stretching at 293
1505cm-1 and 1449cm-1respectively, and C=CH aromatic stretching at 756cm-1. 294
These data corresponds to the functional groups present in BDMCA. These data are 295
similar to those observed by Naama et al. (2010). 296
In the FTIR spectrum of BDMCA-CS nanocomposite, the peak corresponding to OH 297
stretching has broadened (3456cm-1). This may be due to the interaction of BDMCA 298
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with the CS nanocomposite by means of hydrogen bonding. After the loading of 299
BDMCA into the CS nanocomposite, a shift in the peak from 1654cm-1 and 1382cm-300
1to 1626cm-1and 1378cm-1respectively, was observed. It was also interesting to note 301
that a similar range of peak shifts has already been reported by Das et al. (2010)302
suggesting the possibility of encapsulation of curcumin into the CS nanocomposite. 303
Thus, it is logical to infer that the peak shift observed in our study signals that the 304
drug BDMCA might have got encapsulated in the CS nanocomposite. Further, some 305
extra peaks corresponding to the composite compound starch and the drug, BDMCA 306
also appeared in the FTIR spectrum of BDMCA-CS. They are: (i) peaks at 1097cm-1 307
and 887cm-1 due to C-O-C symmetrical stretching of starch (ii) the peak at 756cm-1308
(C=C-H aromatic stretching), 1451cm-1 (C=C aromatic stretching) and 1239cm-1309
(C=O stretching) are the characteristic peaks of BDMCA. These peaks are supportive 310
data for confirming the presence of our drug BDMCA within the CS nanocomposite. 311
For the most part, the FTIR spectrum of BDMCA-CS nanocomposite reveals that the 312
drug got encapsulated within the CS nanocomposite.313
314
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315
Figure 3. FTIR Spectrum of Chitosan, Starch, CS nanocomposite, BDMCA and 316BDMCA-CS nanocomposite317
318
3.4 Percentage Drug Content and drug encapsulation efficiency319
The amount of drug per unit volume present in the formulation is the drug content. 320
The amount of drug entrapped within the nanocarrier is its drug encapsulation 321
efficiency. The percentage drug content and drug encapsulation efficiency are were322
calculated with a constant amount of BDMCA (50mg) but varying the ratio of chitosan 323
and starch. The drug content was found to be 25.75%, 17.5% and 24.25% for the 324
formulations BDMCA-CS 3:1, 1:1 and 1:3 respectively. The encapsulation efficiency 325
was found to be 88.98%, 86.54% and 86.60% for the formulations BDMCA-CS 3:1, 326
1:1 and 1:3 respectively. Both the parameters showed maximum values in the 327
formulation BDMCA-CS 3:1. This may be attributed to the increased amount of 328
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chitosan since longer chain of chitosan can encapsulate larger amount of BDMCA in 329
the presence of TPP as a cross linking agent. Only minor difference was observed in 330
the encapsulation efficiency between BDMCA-CS 1:1 (86.54%) and 1:3 (86.60%), 331
though the proportion of Starch was higher in the formulation BDMCA-CS 1:3. The 332
second leading drug loading capacity was observed in BDMCA-CS 1:3 (24.25%). 333
This may be due to the higher quantity of Starch. BDMCA-CS 1:1 (17.5%) had the 334
least drug loading capacity as the shorter chains of Chitosan and Starch can load the 335
minimum amount of BDMCA. We therefore interpret that the percentage drug content336
and drug encapsulation efficiency increases with increasing concentration of the 337
polymer and the increase was more predominant with increasing concentration of 338
chitosan.339
3.5 In-vitro drug release studies340
From our results discussed so far, it is obvious that the formulations BDMCA-CS 3:1 341
and 1:3 exhibit higher percentage drug content and drug encapsulation efficiency. 342
We therefore used these two formulations for in-vitro drug release studies. The drug 343
(BDMCA) release profile from the two formulations (BDMCA-CS 3:1 and 1:3) are 344
shown in Figure.4 and 5 respectively. The drug release profile showed a very slow 345
release of BDMCA from the two different formulations for every one hour. 346
Formulation BDMCA-CS 3:1 showed about 20% of the drug release in 10 hours, 35% 347
of drug release in 20 hours and about 40% in 24 hours. Whereas, the formulation 348
BDMCA-CS 1:3 showed about 23% drug release in 10 hours, 39% in 20 hours and 349
45% release in 24 hours. There was no initial burst release found in both the 350
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formulations. Though there was only minor difference between the release profiles of 351
the two formulations, the formulation BDMCA-CS 3:1 showed a very slow release 352
compared to BDMCA-CS 1:3. One of the primary criteria for passive targeting of 353
cancer cells is that the drug delivery system should be capable of retaining the drug 354
for a prolonged period of time, thus, facilitating a controlled and sustained release of 355
the drug without any initial burst release (Das et al., 2010). Our results fulfill this 356
criterion as we also observed a controlled release of BDMCA without any burst 357
release. Thus, we expect that the chitosan-starch (CS) nanocomposite can be a 358
perfect drug delivery tool for cancer cells. Therefore, we subsequently investigated 359
the ability of CS nanocomposite particles to release BDMCA into breast cancer cells 360
possibly via passive tumor targeting. Sustained drug release may be the reason for 361
delayed saturation, as ob served by the absence of plateu in figures 4 and 5. The 362
mechanism of drug release may be due to diffusion or polymer dissolution as 363
explained in the next section. 364
365
Figure 4. In-vitro release profile of bis-demethoxy curcumin analogue (BDMCA) from 366
the formulation with chitosan: starch in the ratio of 3:1367
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368
Figure 5. In-vitro release profile of bis-demethoxy curcumin analogue (BDMCA) from 369
the formulation with chitosan: starch in the ratio of 1:3370
3.6 Drug release kinetics371
The release profile, i.e., the order and the mechanism of BDMCA release from the 372
nanocomposite can be interpreted by different kinetic models such as, Zero order 373
model, First order model, Higuchi’s model, Korsmeyer-Peppas model and Hixson-374
Crowell model. Therefore, the kinetic release data of BDMCA-CS 3:1 (Figure.6a) and 375
1:3 (Figure.6b) were fitted into the Zero order model, First order model, Higuchi’s 376
model, Korsmeyer-Peppas model and Hixson-Crowell model. 377
The First order plot exhibited good linearity (R2= 0.992 for both the formulations, 378
BDMCA-CS 3:1 and 1:3) when compared to Zero order plot (R2= 0.984 and 0.977 for 379
BDMCA-CS 3:1 and 1:3, respectively). This shows that the amount of BDMCA 380
released is dependent on the concentration of the drug loaded in the nanocomposite 381
i.e., drug release is concentration dependent (Cho et. al., 2008).382
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We also observed good linearity in Higuchi plot (R2= 0.937 and 0.964 for BDMCA-CS 383
3:1 and 1:3, respectively). This observation clearly indicates that the drug release is 384
due to diffusion mechanism. 385
386
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387
Figure 6. Release kinetics of BDMCA from the formulation a) BDMCA-CS 3:1 b) 388
BDMCA-CS 1:3389
The release of BDMCA from the CS nanocomposite exhibited a good fitting with 390
Hixson-Crowell plot with a good linearity value (R2= 0.990 and 0.988 for BDMCA-CS 391
3:1 and 1:3, respectively). This shows that the BDMCA may be released due to 392
polymer dissolution or erosion (Rahman et al., 2011).393
The value of n in Korsmeyer-Peppas plot is 1.070 and 1.017 for BDMCA-CS 3:1 and 394
1:3, respectively. Dash et al., (2010) have suggested that an>0.5 corresponds to 395
Fickian diffusion mechanism, 0.45<n<0.89 corresponds to non-Fickian mechanism, 396
n=0. 89 to relaxational transport (Case II) while n>0.89 refers to Super case II 397
transport. Thus, we suggest that the release of BDMCA form CS nanocomposite is 398
Super case II transport.399
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3.7 Anticancer assay400
Anticancer agents exert their activity by blocking the cell cycle progression and 401
triggering apoptotic cell death. The half-maximal inhibitory concentration (IC50) is the 402
concentration of the anticancer agent that induces 50% of the cell death. The 403
anticancer activity of BDMCA-CS nanocomposite particles was determined by 404
performing MTT assay on MCF-7 and Vero cell lines after incubating these cells with 405
different concentration of BDMCA-CS nanocomposite particles.406
The MCF-7 and Vero cells were treated with different concentrations of BDMCA-CS 407
nanocomposite particles (1.953, 3.906, 7.8125, 15.625, 31.25, 62.5, 125, 250, 500 408
and 1000 µg/ml). Microscopic images of BDMCA-CS treated and untreated (Control) 409
MCF-7 and Vero cell lines are shown in Figure.7a & 7b. The microscopic images 410
revealed that the BDMCA-CS had a greater cytotoxic effect on MCF-7 cells than on 411
Vero cells. MCF-7 cells treated with higher concentration of BDMCA-CS exhibited 412
morphological changes such as membrane shrinkage, cell aggregation, detachment 413
from the well surface and rounding up of cells. These morphological features indicate 414
the BDMCA-CS induced cell death (Vijayarathna & Sasidharan, 2012).415
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416
Figure 7a. Morphology of MCF-7 cancer cells with BDMCA-CS 417nanocomposite particles418
(i) Control419(ii) Cells treated with 62.5µg/ml of BDMCA-CS 420(iii) Cells treated with 250µg/ml of BDMCA-CS421(iv) Cells treated with 1000 µg/ml of BDMCA-CS422
423
424
Figure 7b. Morphology of VERO cells treated with BDMCA-CS nanocomposite 425particles426
(i) Control 427(ii) Cells treated with 62.5µg/ml of BDMCA-CS428(iii) Cells treated with 250µg/ml of BDMCA-CS 429(iv) Cells treated with 1000 µg/ml of BDMCA-CS430
431
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A cell is said to be viable when the key enzyme, dehydrogenase present in the 432
mitochondria of the cell, converts the yellow colored MTT dye into purple colored 433
formazan crystals. Thus, the absorbance reading at 594 nm indicates the number of 434
viable cells. The cell viability of MCF-7 and Vero cells against increasing 435
concentration of BDMCA-CS nanocomposite particles are graphically represented in 436
Figure.8. 437
438
Figure 8. Cell viability of MCF-7 and VERO cells at increasing concentration of 439
BDMCA-CS nanocomposite440
4. DISCUSSION 441
We suggest that the size of the nanocomposite particles increases with increasing 442
concentration of at least one of the polymers (320 to 91nm). It has been reported that 443
the size of the nanoparticles used in drug delivery applications should be large 444
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enough (100 to 600nm) to prevent their leakage into blood capillaries but small 445
enough (150 to 200nm) to escape the reticuloendothelial system (Shin et al., 2008). 446
This context is related to enhanced permeability and retention (EPR) effect. It is the 447
property by which certain sizes of molecules typically liposomes, nanoparticles, and 448
macromolecular drugs tend to accumulate in tumor tissue much more than they do in 449
normal tissues. Therefore, we infer that all the three formulations prepared by us 450
may further be analyzed for drug loading and drug encapsulation efficiency. Previous 451
studies suggested that the spherical nanoparticles are best suited for drug delivery 452
applications because they are capable of overcoming the limitations such as poor 453
drug loading capacity, low drug efficacy, broad size distribution and non-specific drug 454
toxicity (Shin et al., 2008). These findings coincide with our results, as the particles 455
synthesized by us were also spherical. Thus, we suggest that the BDMCA loaded 456
CS nanocomposites are good candidates for drug delivery. The FTIR data of 457
chitosan, starch and CS nanocomposite confirms that the spherical particles 458
(observed in SEM) are nanocomposite of chitosan and starch, gelated with TPP. 459
Among the three ratios investigated, the percentage drug content and the drug 460
encapsulation efficiency were found to be highest for the composite with highest461
proportion of chitosan. 462
First order, Korsmeyer-Peppas & Hixson-Crowell Kinetics plot of our study reveals a 463
good linearity. Previous studies on sustained release of Ranolazine (Alam et al., 464
2011) suggested that good linearity value in vitro model is an indicator of sustained 465
drug release by both diffusion and polymeric erosion. These findings are in line with 466
our reports revealing the dual mechanism involved in the release of BDMCA from CS 467
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nanocomposite. Thus we could suggest that CS nanocomposite may help with468
sustained release of BDMCA, which may be useful for many therapeutic applications. 469
With these findings as a preliminary approach, we carried out cytotoxic assay (in 470
Vero and MCF-7) to check the anticancer activity of CS-BDMCA. This may be a 471
proof for the sustained release of BDMCA. 472
On comparing the cell viability of Vero and MCF-7 cells, it can be observed that the 473
cell viability of the MCF-7 cells decreased at a higher rate as the concentration of the 474
BDMCA-CS increased. Thus, it is obvious that BDMCA-CS functions as an 475
anticancer agent by inducing cell death via cytotoxicity. Further, it was found that 476
62.5µg/ml concentration of BDMCA-CS induced 50% of the MCF-7 cells (IC50), 477
whereas more than 250µg/ml concentration of BDMCA-CS induced 50% cell death in 478
Vero cells.479
5. CONCLUSION480
For the first time, we have formulated BDMCA loaded chitosan-starch nanocomposite 481
for the controlled release of BDMCA. The formulation BDMCA-CS 3:1 showed good 482
drug entrapment efficiency and percentage drug content, followed by BDMCA-CS 483
1:3. These formulations showed sustained release of BDMCA. Subsequently,484
formulation BDMCA-CS 3:1 showed anticancer activity against the breast cancer cell 485
lines (MCF-7). Release kinetics studies revealed that the release of BDMCA from the 486
CS nanocomposite takes place by both diffusion and polymeric erosion. On the 487
whole, we speculate that the CS nanocomposite of ratio 3:1 can be a better delivery 488
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tool for BDMCA to treat breast cancer. The effect of this formulation of other cancer 489
models is underway in our laboratory.490
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iptHighlights
BDMCA loaded chitosan - starch (BDMCA-CS) nanocomposite was developed by ion gelation method
BDMCA:CS (3:1 ratio) nanocomposite shows highest entrapment efficiency and percentage drug content
BDMCA-CS nanocomposite exhibits anticancer potential against MCF-7 breast cancer cell line and non
toxic to VERO cell lines.
BDMCA-CS released the drug in a controlled fashion due to diffusion and polymeric erosion.
![Nanocomposite [5]](https://img.pdfslide.us/doc/110x75/577c7ecf1a28abe054a26499/nanocomposite-5.jpg)
