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Title: “Biodegradable Chitosan Nanogels Crosslinked withGenipin”
Authors: Maite Arteche Pujana, Leyre Perez-Alvarez, LuisCarlos Cesteros Iturbe, Issa Katime
PII: S0144-8617(13)00128-8DOI: doi:10.1016/j.carbpol.2013.01.082Reference: CARP 7426
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Received date: 13-11-2012Revised date: 14-1-2013Accepted date: 15-1-2013
Please cite this article as: Pujana, M. A., Perez-Alvarez, L., Iturbe, L. C. C., & Katime, I.,“Biodegradable Chitosan Nanogels Crosslinked with Genipin”, Carbohydrate Polymers(2010), doi:10.1016/j.carbpol.2013.01.082
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CARBPOL-D-12-02432-R1. 1
“Biodegradable Chitosan Nanogels Crosslinked with Genipin”2
3
Maite Arteche Pujana, Leyre Pérez-Álvarez, Luis Carlos Cesteros Iturbe, Issa 4
Katime.5
6
Grupo de Nuevos Materiales y Espectroscopia Supramolecular. Departamento 7
de Química Física. Facultad de Ciencia y Tecnología. Universidad del País 8
Vasco (UPV/EHU). 644 Bilbao, Spain.9
E-mail: [email protected]
+3494601271111
12
Keywords13
Genipin, nanogels, chitosan.14
15
ABSTRACT16
Chitosan nanoparticles crosslinked with genipin were prepared by reverse 17
microemulsion that allowed to obtain highly monodisperse (3-20 nm by TEM) 18
nanogels. The incorporation of genipin into chitosan was confirmed and 19
quantitatively evaluated by UV-Vis and 1H NMR. Loosely crosslinked chitosan 20
networks showed higher water solubility at neutral pHs than pure chitosan. The 21
hydrodynamic diameter of the genipin-chitosan nanogels ranged from 270 to 22
390 nm and no remarkable differences were found when the crosslinking degree 23
was varied. The hydrodynamic diameters of the nanoparticles increased slightly 24
at acidic pH and the protonation of ionizable amino groups with the pH was 25
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confirmed by the zeta potential measurements. The biocompatible and 26
biodegradable nature, as well as the colloidal and monodisperse particle size of 27
the prepared nanogels, make them attractive candidates for a large variety of 28
biomedical applications.29
30
HIGHLIGHTS31
32
Chitosan was crosslinked with genipin in reverse microemulsion medium.33
Chitosan-genipin crosslinking reaction was quantitatively evaluated by UV-Vis 34
and 1H NMR.35
Chitosan-genipin nanogels showed improved water solubility at neutral pHs.36
Swelling behaviour of the nanogels was studied as function of crosslinking 37
degree and pH.38
39
Keywords40
Genipin, nanogels, chitosan.41
42
43
INTRODUCTION44
Chitosan is a linear and partly acetylated (1-4)-2-amino-2-deoxy-β-D-45
glucan isolated from marine chitin (Muzzarelli, Boudrant, Meyer, Manno,46
DeMarchis & Paoletti, 2012; Muzzarelli, Jeuniaux, Gooday, 1986). In contrast to 47
other polymers, chitosan is positively charged due to the protonation of its 48
primary amines weak basic groups, which give it special characteristics and 49
feasibility to chemical modification. Chitosan exhibits many favourable 50
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characteristics such as biodegradability, nontoxicity and biocompatibility, which 51
give it a great potential in a wide range of areas such as food, cosmetics,52
wastewater treating, biotechnology, or biomedicine (Shahidi, Arachchi & Jeon, 53
1999; Ravi Kumar, 2004; Crini, 2005)54
Chitosan has been extensively studied for delivery of bioactive reagents, 55
including drugs (Felt, Buri & Gurny, 1998), proteins and genes (Malhotra, 56
Kulamarva, Sebak, Paul, Bhathena, Mirzaei & Prakash, 2009) and it is, in this 57
respect, one of the preferred candidates in advanced fields like gene delivery58
(Liu & Yao, 2002), transnasal delivery (Illum, Farraj & Davis, 1994) or ocular 59
diseases treatment (Alonso & Sánchez, 2003). Regarding this purpose chitosan 60
has been prepared in a wide variety of forms, namely hydrogels, beads, films, 61
tablets, microspheres, composites, micro and nanoparticles (Miyazaki,62
Nakayama, Oda, Takada & Attwood, 1994; Lee, Nam, Im, Park, Leeb, Seol, 63
Chung & Lee, 2002; Banerjee, Mitra, Kumar Singh, Kumar Sharma & Maitra, 64
2002).65
Nanoparticles have a special role in targeted drug delivery due to their 66
long shelf life, easy transport to different body sites and high drug entrapment 67
efficacy (Agnihotri, Mallikarjuna & Aminabhavi, 2004). Chitosan nanoparticles 68
can be obtained by chemical crosslinking leading to quite stable matrixes. These 69
chitosan nanogels are commonly prepared by modification with bifunctional 70
agents such as diisocyanate, diepoxy compounds, dialdehyde and other 71
reagents (Wenling Mingyu, Qiang, Yandao, Nanming & Xiufang, 2005; Rinaudo, 72
2010). Among them, in the last decades glutaraldehyde was the most broadly 73
used crosslinker agent in covalent formulations. However, due to the proved 74
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toxicity of glutaraldehyde, recently, biocompatible crosslinking agents have 75
received much attention in the field of biomedical application.76
Extended studies about genipin, an alternative natural crosslinking agent, 77
has shown its ability to form biocompatible and stable crosslinked products78
10,000 times less cytotoxic than glutaraldehyde (Sung, Huang, Huang & Tsai, 79
1999). Genipin is an iridoid glucoside extracted from Gardenia that has been 80
used in traditional Chinese medicine and as a blue colorant in the food industry.81
Genipin reacts under mild conditions with primary amine groups and this 82
reaction, which is undoubtedly identified by mean of its inherent blue coloration, 83
has been employed to crosslink chitosan (Jin, Song & Hourston, 2004) and 84
other amino group containing biomaterials (González, Strumia & Alvarez 85
Igarzabal, 2011).86
Genipin has been widely employed in the field of biomaterials specially in 87
studies of tissue fixation and regeneration in various biomaterials and natural 88
biological tissues, or in studies of drug delivery of macrogels, microspheres and 89
semi-interpenetrating polymer networks (semi-IPNs) (Muzzarelli, Greco, 90
Busilachi, Sollazzo & Gigante, 2012). Additionally, genipin has been employed 91
to obtain nanocomposites consisted of metal nanoparticles embedded in 92
chitosan crosslinked matrixes (Liu & Huang, 2008). However, there are not 93
many publications relating to genipin crosslinked nanoparticles (Choubey &94
Bajpai, 2010; Maggi, Ciccarelli, Diociaiuti, Casciardi & Masci, 2011) which95
encourages us the use of genipin as a crosslinking agent to develop chitosan 96
nanogels for biomedical applications.97
Since size distribution plays an important role in drug delivery application, 98
it is necessary to prepare uniformly sized nanoparticles. However, the size 99
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distribution of chitosan nanoparticles is very difficult to control. Various studies 100
have shown that reverse microemulsion (w/o) is an effective way to prepare low 101
sized particles with uniform particle size distribution. This promising method for 102
the effective preparation of chitosan nanoparticles is a transparent, isotropic and 103
thermodynamically stable synthesis medium. Microemulsions not only act as 104
microreactors for the formation of nanoparticles but also inhibit excessive 105
aggregation of the forming particles (Liu, Shao, Gea & Chena, 2007). Several 106
authors have employed the well stablished microemulsion formulation based on 107
cyclohexane/Triton X100/n-hexane/water for obtaining chitosan nanosized 108
particles (Wang, Wang, Luo & Dai, 2008; Arteche Pujana, Pérez-Alvarez, 109
Cesteros Iturbe & Katime, 2012).110
In this work chitosan and genipin were chosen for preparing polymeric 111
nanogels by crosslinking of chitosan. Both of these compounds display 112
advantageous biomedical properties. Nanogels were preparing by w/o 113
microemulsion method and their physicochemical properties, such as, particle 114
size, pH-sensitivity swelling, surface charge or water solubility, were studied.115
116
EXPERIMENTAL117
118
Materials119
Low molecular weight chitosan (Aldrich) was purified using the procedure 120
previously described (Signini & Campana, 1999). The deacetylation degree of 121
chitosan determined by 1H NMR was 79% which is in good agreement with the 122
value reported by the supplier (75-85%). Its viscosity average molecular weight 123
of chitosan measured by an Ubbelohde capillary viscometer (HAc 0.3M/NaAc124
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0.2M, 25ºC) (Rinaudo, Milas & Dung, 1993) was 66,000 g/mol. Genipin, triton X-125
100 and hexanol (for synthesis, 98%) were purchased from Sigma-Aldrich. 126
Cyclohexane (for synthesis, 98%), acetic acid (for analysis, 99.8%) and ethanol 127
(for analysis, 96%) were supplied from Panreac and D-glucosamine 128
hydrochloride by Acros Organics.129
130
Preparation of nanogels131
1g of chitosan was dissolved in 100 mL of 1%v/v aqueous acetic acid 132
solution and separately genipin was dissolved in distilled water (1-540 mg/mL). 133
Chitosan nanoparticles were prepared using a procedure that was 134
previously described by Jia et al. (Jia, Yujun & Guangsheng, 2005) with minor 135
modification. Two separate W/O microemulsions were produced. The first 136
microemulsion (MC) was formed by dropwise addition of Triton X-100 to a 137
mixture of cyclohexane: n-hexanol: chitosan solution in ratio of 2.75:1:1 138
respectively. Triton X 100 was added until microemulsion became optically 139
transparent. The second microemulsion (MG) containing genipin solution but 140
without chitosan was prepared with the same procedure. Then, MG 141
microemulsion was added to MC microemulsion under magnetic stirring at 142
different crosslinker stoichiometric ratio. The crosslinking reaction was carried 143
out at 25 ºC for 4 days. During crosslinking reaction the microemulsion became 144
bluish. The microemulsion was broken by adding ethanol to obtain blue colored 145
nanoparticles. Nanogels were washed repeatedly with ethanol by centrifugation 146
(9000rpm, 10 min, at room temperature). Finally prepared nanogels were 147
washed with distilled water, ultrafiltered (OMEGA 76 MM 100K 12/PK) and 148
vacuum dried (55ºC, 24 hours).149
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150
Methods151
The nanogels were analyzed with NMR spectroscopy. 1H spectra were152
obtained on a Bruker Avance 500 MHz instrument and the samples were 153
dissolved in 2% w/w. CD3COOD/ D2O. Solid samples were analyzed by 13C 154
NMR spectroscopy with a Bruker Avance III 400 spectrometer.155
A Philips CM120 with Olympus SIS Morada digital camera transmission 156
electron microscope was used to characterize the size and morphology of the 157
dried chitosan nanoparticles. Nanoparticles were prepared dissolving dried 158
powder in 1%v/v aqueous acetic acid solution with a concentration of 1mg/mL.159
which was then dried in a vacuum chamber.160
UV–Vis spectroscopy measurements were registered in a Cintra 303 UV-161
Vis Spectrophotometer. 162
The genipin-chitosan crosslinking reaction was characterized by UV-Vis. 163
Along the crosslinking reaction, spectra of the microemulsion samples were 164
obtained at different time intervals in a wavelength range from 200 to 700 nm. 165
Furthermore, the quantitative determination of the reacted free amino groups in 166
nanoparticles crosslinked with genipin was carried out by mean of the previous167
calibration with D-glucosamine in the microemulsion medium. D-glucosamine 168
(50 to 750 mg/L) and genipin (500 mg/L) after being dissolved in the described 169
microemulsion medium were incubated at 25ºC. The absorbance at 605 nm was 170
measured and the obtained linear regression equation was A = 0.0188+ 171
0.0021X (R2 = 0.99764).172
UV-Vis spectroscopy was also employed for the study of the water 173
solubility of the nanogels at different pH values by mean of the measurement of 174
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the transmittance of the nanogels dispersions at 750 nm. Nanogels were 175
dissolved in 1% HAc solution (1 mg/5 mL), and the transmittance was measured 176
24 hours after the pH of the solution was adjusted by the addition of 2 N NaOH 177
solution.178
Dynamic laser light scattering measurements for determining the size of 179
the nanogels were performed using a Brookhaven BI-9000AT with a goniometer. 180
A water-cooled argon-ion laser was operated at 514.5 nm as the light source. 181
The time dependence of the intensity autocorrelation function of the scattered 182
intensity was obtained by using a 522 channel digital correlator. The size of the 183
nanoparticles was determined from the diffusion intensity of the particles using 184
the Stokes-Einstein equation. The size distribution was obtained by NNLS 185
analysis and the uncertainties of the measurements represented the standard 186
deviation of the mean of the replicate runs. The dried powder samples were 187
dispersed in 1% HAc solution (1mg/mL) during 3 days and then diluted until the 188
concentration was 40 mg/L. All measurements were made at room temperature.189
The pH was changed with 2 N NaOH solution.190
X-ray diffraction patterns were obtained by a Transmision STOE Stadip 191
X-ray diffractometer with a goniometer speed of 0.1º/90s. The range of 192
diffraction angle 2 was 2.5-34º.193
Electrophoretic mobility measurements were performed with a Zeta-Sizer 194
IV (Malvern Instruments). Nanogels were dispersed in 1% HAc solution195
(1mg/mL) during 3 days and finally diluted to a concentration of 40 mg/L. Data 196
were obtained from the average of at least ten measurements in universal cells. 197
The external pH was varied by adding 2 N NaOH solution.198
199
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RESULTS AND DISCUSSION200
201
202
1.- Study of the crosslinking reaction203
204
Covalently crosslinked chitosan nanoparticles were prepared within a 205
reverse microemulsion medium in order to control the colloidal particle size of 206
the prepared nanogels. This modification reaction took place when separately 207
prepared chitosan and genipin microemulsion were mixed as described above. 208
This crosslinking occurs by mean of a complex mechanism which is believed 209
that consists in two reactions involving different sites on the genipin molecule 210
(Butler, Ng & Pudney, 2003) and produces dark-blue coloration in the reaction 211
mixture. The appearance of this coloration in the reaction mixture of genipin and 212
chitosan is traditionally considered as rapid evidence that the crosslinking 213
reaction is taking place (Butler, Ng & Pudney, 2003; Muzzarelli, 2009).214
The evolution of the crosslinking reaction was monitored by mean of the 215
UV-Vis spectra of the final microemulsion. As can be seen in Figure 1A, a new 216
peak appeared at 605 nm, which corresponds to the formed blue pigment, and 217
continued to increase in intensity throughout the course of the reaction. 218
Ramos et al. (Ramos-Ponce, Vega, Sandoval-Fabián, Colunga-Urbina, 219
Balagurusamy, Rodriguez-Gonzalez & Contreras-Esquivel, 2010) described an 220
efficient colorimetric method using genipin as a specific reagent for quantitative221
determination of free amino groups of chitin derivatives. Following this 222
procedure, the relationship between the absorbance of the microemulsion at 605 223
nm and the concentration of free amino groups in the nanogels was determined 224
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by mean of a calibration curve with standard solutions of D-glucosamine. The 225
method reported by Ramos et al. could be applied to the microemulsion medium 226
after slight modifications. The concentration of modified free amino groups as a 227
function of reaction time was analyzed in order to optimize the reaction time.228
Results of this study are shown in Figure 1B, demonstrating that after 7 days the 229
end point of the reaction was reached. 230
231
3.- NMR characterization232
233
The success of the modification reaction was also confirmed and 234
quantitatively evaluated by 1H NMR. Pure chitosan shows a peak at 2.0–2.1 235
ppm corresponding to the three protons of N-acetyl glucosamine (GlcNAc) and a 236
peak at 3.1–3.2 ppm due to H-2 proton of glucosamine (GlcN) residues which 237
represent the primary amino group content of the chitosan. Figure 2 displays the 238
spectra of several genipin-chitosan nanogels. A clear decrease of the signal at 239
3.2 ppm was observed for all the samples, indicating the loss of amine groups 240
by reaction with genipin showing that the addition of genipin to chitosan was 241
successfully carried out.242
The peak of the 1H NMR spectrum of chitosan corresponding to the 243
methyl protons at 2.0 - 2.1 ppm possess the highest resolution (Kasaai, 2010)244
and was used for the determination of the free amino content by mean of the245
integration of the resonance of H-2 protons at 3.2 ppm. Thus, the modification 246
degree of the chitosan chains was evaluated by the decrease of the 247
glucosamine signal (3.2 ppm). The crosslinking grade was calculated assuming 248
that the reaction occurs on both reaction sites of the genipin molecule. Table 1 249
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lists the nanogels compositions obtained by 1H NMR analysis for each initial 250
reaction feed. The data collected in Table 1 show a rather good correlation 251
between genipin stoichiometric ratio (with respect to the total amount of 252
glucosamine units) in initial reaction mixture and final modification degree in the 253
nanogels, especially for intermediate compositions. These results suggest that 254
the linkage genipin-chitosan occurs in a ratio around 1:2 and therefore the self-255
polymerization of genipin is not likely to take place. However, for high crosslinker 256
content, the ratio genipin feed: modified chitosan increased. Assuming that the 257
reaction yields did not varied significantly, it could be related to a higher258
probability that polymerization takes place. This in accordance with an 259
intermediate brownish colour appeared during crosslinking reaction of these 260
samples before the blue intense coloration (Mi, Shyu & Peng, 2005).261
On another hand, modification degrees of chitosan in the nanogels 262
obtained by 1H NMR analysis agreed with those obtained by UV-Vis 263
spectroscopy (Table 1).264
Besides, 13C NMR spectroscopy also corroborates the modification of 265
linear chitosan with genipin. Figure 3 shows the 13C NMR of initial pure chitosan 266
and chitosan crosslinked with genipin. The crosslinking of chitosan with genipin267
results in some slight changes on the 13 C NMR spectrum of chitosan. The 268
resonance signal of chitosan C-4 at 81-87 ppm decreased and becomes into a 269
shoulder. Mi et al. (Mi, Sung & Shyu, (2000); Mi, Sung & Shyu, 2001) attributed 270
this fact to a conformational change from the linear structure of original chitosan 271
to crosslinked chitosan, revealing the formation of a secondary amide and 272
heterocyclic amino linkage, and so, the crosslinking of chitosan. Additionally, a273
slight shift of C-1 resonance from 112 ppm to a broad resonance centered at 274
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around 109 ppm occurs, which is attributed to the incorporation of the 275
heterocyclic ring at C-2 (Mi, Sung & Shyu, 2000).276
277
4.- Water solubility 278
The applications of chitosan nanoparticles suffer severe limitations 279
because they are no dispersible in neutral or alkaline pH aqueous media. This 280
water insolubility in high pH solutions is due to the presence of rigid crystalline 281
domains, formed by intra- and/or intermolecular hydrogen bonding (Nishimura, 282
Kohgo, Kurita & Kuzuhar, 1991).283
The solubility of the prepared genipin-chitosan nanogels was 284
characterized as a function of pH and compared with the unmodified chitosan. 285
Figure 4A shows the transmittance at 750 nm of aqueous solutions of linear 286
chitosan and genipin-chitosan nanogels prepared in the above described 287
conditions. 288
In general, as the pH decreases the protonation of free amino groups of 289
chitosan chains takes place and the H-bonding interactions decreases, 290
improving water-polymer interaction and consequently increasing the 291
transmittance of aqueous dispersions (Figure 4A). So, when pH increases the 292
dispersions become opaque and particles aggregation takes places. 293
The solubility of the nanogels was determined from pH50 parameter that is 294
defined as the pH value when the transmittance at 750 nm reached 50%. 295
Loosely crosslinked nanogels (< 7.5 mol. % genipin) showed a slightly improved 296
stability with the pH, displaying a value of 7.3 for the pH50 parameter, while the 297
value measured for pure chitosan was 7.0. When the genipin content was higher 298
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than 7.5 mol. % the pH50 of the nanogels was lower than that of the pure 299
chitosan in all the cases.300
It is well kwon that the modification of chitosan with more or less 301
bulky pendant groups leads to the reduction of hydrogen bonding in 302
chitosan and therefore, the solubility of the chitosan derivatives increases. In this 303
regard, the increase in water solubility of PEGylated chitosan (Chan, Kurisawa, 304
Chung & Yang, 2007) has been attributed to the decrease of intermolecular 305
interactions, as well as the swelling of N-alkyl and N-acylated chitosans in 306
water, in spite of their hydrophobicity (Ravi Kumar, 2000; Shasiwa, Kawasaki, 307
Nakayama, Muraki, Yamamoto & Aiba, 2002).308
In general, chitosan derivatives that precipitate in basic medium have a 309
high degree of crystallinity. On the contrary, chitosan obtained with amorphous 310
form and loosely aggregated state is soluble in water (Lu, Song, Cao, Chen & 311
Yao, 2004).312
Figure 4B shows the X-ray diffractograms of some of the samples whose 313
solubility behaviour is displayed in Figure 4A. Pure chitosan sample displays the 314
typical pattern of chitosan substrates with peaks at 2 around 11 and 20º. As 315
can be observed, the chemical crosslinking with genipin causes the loss of 316
crystallinity in the samples. All the studied genipin-chitosan nanogels displayed 317
a difractogram of an amorphous material. 318
Thus, it could be concluded that a moderate crosslinking disturbs the intra 319
and inter intermolecular hydrogen bonding resulting in the observed 320
enhancement in solubility. However, as crosslinking increases (up to 7.5 mol. 321
%), in spite of the weakened intra-intermolecular interactions of chitosan chains, 322
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the nanogels become more and more compact and a close and rigid network is 323
generated, leading to a substantial reduction of the water solubility.324
325
5.- Particle size and swelling properties of the nanogels326
327
The morphology and particle size distribution of dried nanogels was 328
studied by TEM. TEM images (Figure 5A) indicate that genipin-chitosan 329
nanogels are spherical, have nearly uniform particle size distribution (Figure 5B)330
and there is no severe particle agglomeration, which are very interesting 331
qualities for biomedical applications, such as drug delivery. The average 332
diameters of nanoparticles ranged from 3 to 20 nm.333
The particle size of swollen nanogels was determined by QELS334
measurements. Due to the adhesive nature of chitosan in aqueous solution, 335
these nanoparticles tend to form aggregates that lead to a higher average 336
hydrodynamic diameter than the actual size of the particles (Wu, Zhou & Wang, 337
1995). The progressive dilution of the nanoparticle dispersions has been 338
reported as an effective way to reduce the inter-particle interaction and the 339
presence of large aggregates of chitosan is (Sorlier, Rochas, Morfin, Viton &340
Domard, 2003) and was applied in this study. However, the solubility study 341
revealed in comparison with pure chitosan a higher tendency of genipin-chitosan 342
nanogels to aggregate, except for loosely crosslinked ones (7.5 mol. %).343
Figure 6 shows the average diameters of the nanogels determined by 344
QELS at pH = 4.0 for several genipin stoichiometric ratios. No strong correlation 345
was found between the hydrodynamic diameter of the genipin-chitosan nanogels 346
and the crosslinking degree. Besides, as is shown in Figure 7, a slight pH-347
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responsive behaviour was observed. Thus, it could be concluded that the 348
nanogels display a weak swelling capacity due to the rigid and hydrophobic 349
nature of the employed crosslinker.350
The swelling effect induced in an ionic network, such as chitosan 351
nanogels, can be considered as the accumulative effect of the mixing, elastic 352
and ionic contributions (Flory, & Rehner, 1943). In the case of the genipin-353
chitosan nanogels at pH = 4, an increase in crosslinking has opposite effects. 354
On the one hand leads to the diminishing of ionic contribution due to the 355
decrease of ionizable groups in the network, and to the enhancement of the 356
elastic force. Both of these effects tend to limit the swelling of the gel. However, 357
as crosslinker concentration increases (up to 7.5 mol. %), the mixing 358
contribution is drastically reduced, due to the hydrophobic and closed nature of 359
the network, and aggregation takes place, increasing the measured 360
hydrodynamic diameter. This may explain that the determined particle sizes of 361
the nanoparticles varying the content of genipin were fairly similar each other362
(Figure 6).363
At constant degree of crosslinking, when the external pH was varied it 364
was found that the nanoparticles swell slightly at pH below 5.5 (Figure 7). This 365
may be due to the protonation of free amine groups of chitosan chains at low 366
pH, which results in an electrostatic repulsion charge favouring the penetration 367
of water into the gel. For basic pHs the precipitation of the nanogels takes place 368
and particle size or could not be measured or increased as consequences of 369
particle aggregation. Studies of the pH-responsive swelling of genipin-chitosan 370
macrogels also did not displayed an abrupt phase transition (Kaminski, K., 371
Zazakowny, K., Szczubiałka, K., Nowakowska M., 2008) that was observed in372
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other studied chitosan networks (Arteche Pujana, Pérez-Alvarez, Cesteros 373
Iturbe & Katime, 2012). In this sense, Jharen et al. (Jahren, Butler, Adams &374
Cameron, 2010) found that the pH-responsive phase transition decreased with 375
crosslinking in genipin-chitosan hydrogels and disappeared completely for a 376
genipin percentage of 80 mol. %.377
To obtain information about the electrical state of the ionizable groups 378
responsible of the pH-responsive behaviour of the nanoparticles, zeta potential 379
of genipin-chitosan nanogels was measured as function of genipin content 380
(Figure 6), as well as, of pH (Figure 7). In all the samples the value of the zeta381
potential increased up to 20 mV when the pH increased from 4 to 9. All the 382
nanogels showed positive charge at acidic conditions, so all of them maintained 383
the chitosan mucoadhesive and absorption enhancement potential properties.384
It is shown in Figure 6 that the zeta potential values of protonated 385
networks (pH = 4.0) decreased as the nanogels crosslinking was higher, proving 386
the loss of free amine moiety along the chitosan backbone within the 387
modification with genipin. Regarding pH (Figure 7), as the chitosan chains388
contain free amine pH-ionizable groups, a pH variation modifies the electrical 389
state of the network. The amine primaries groups are protonated in acidic media, 390
and the surface charge is positive and increases along the ionization process 391
occurs. So, the progressive protonation of amino groups as pH decreases was 392
confirmed by measuring the electrokinetic potential of the nanogels.393
394
CONCLUSIONS395
396
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Chitosan low-size nanogels were prepared by the combination of two 397
well-known methods such as, reverse microemulsion and crosslinking by 398
genipin. Nanogels sizes ranging from 3-20 nm determined by TEM, and 270-390 399
nm determined by QELS in aqueous media, could be obtained by this covalent 400
crosslinking method. Loosely crosslinked nanogels displayed an improved water 401
solubility compared with pure chitosan. The variation of water solubility of 402
chitosan due to the crosslinking with genipin could be consider as a compromise 403
between the decrease of crystallinity and the elastic force within the generated 404
network. This elastic contribution may play an essential role in the water 405
solubility and pH-sensitive swelling of the nanogels due to the low molecular 406
weight and rigid nature of genipin molecule.407
408
ACKNOWLEDGEMENTS409
410
Financial support for this work was provided by the Basque Government 411
and the University of the Basque Country (UPV/EHU), for awarding a fellowship.412
413
414
REFERENCES 415
416
Agnihotri, S. A., Mallikarjuna, N. N. & Aminabhavi, T. M. (2004). Recent 417
advances on chitosan-based micro- and nanoparticles in drug delivery.418
Journal of Controlled Release, 100, 5–28.419
Alonso, M. J. & Sánchez, A. (2003). The potential of chitosan in ocular drug 420
delivery. Journal of Pharmacy and Pharmacology, 55, 1451–1463.421
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FIGURE CAPTIONS
FIGURE 1. Evolution of A) the visible absorption spectrum of chitosan
microemulsion during crosslinking reaction with genipin and B) modification
degree of chitosan with crosslinking reaction time for: ○) 7.5, □) 12.5, ●) 18.8, ■)
37.5 mol. % of genipin.
FIGURE 2. 1H NMR spectrum of genipin-chitosan nanogels (7.5 mol. % of
genipin) and amplified resonance signal corresponding to the Ha protons
(chitosan H-2 of GlcN ) of nanogels crosslinked with different genipin feeds (0,
7.5, 15 and 50 mol. %).
FIGURE 3. 13 C NMR spectrum of (A) chitosan nanoparticles crosslinked with
genipin and (B) pure chitosan.
FIGURE 4. A) Transmittance variation with external pH for () pure chitosan and,
genipin-chitosan nanogels with (○) 7.5, (□) 12.5, () 15 and () 25 mol. % of
genipin in feed (1 mg/50ml in 1% HAc solution). B) X-Ray diffraction pattern of
genipin-crosslinked nanoparticles with different mol % of genipin.
FIGURE 5. A)TEM microphotograph of genipin crosslinked chitosan
nanoparticles. (12.5 mol. % of genipin) and, B) Particle size distribution obtained
by TEM.
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FIGURE 6. (□) Zeta potential (●) and Particle size determined by QELS for
genipin-chitosan nanogels at pH = 4.0 as function of crosslinker content.
FIGURE 7. (●) Average hydrodynamic diameter and (○) zeta potential of
nanogels as function of external pH (for 12.5 mol. % crosslinker).
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HIGHLIGHTS
Chitosan was crosslinked with genipin in reverse microemulsion medium.
Chitosan-genipin crosslinking reaction was quantitatively evaluated by UV-Vis
and 1H NMR.
Chitosan-genipin nanogels showed improved water solubility at neutral pHs.
Swelling behaviour of the nanogels was studied as function of crosslinking
degree and pH.
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TABLES
TABLE 1. Modification degree of obtained genipin-chitosan nanogels analyzed by 1H
NMR and UV-Vis spectroscopy.
1H NMR UV-Vis
Genipin Feed
(stoichiometric
mol.%)
Modification
degree
(mol. %)
Crosslinking
(mol. %)
Modification
degree
(mol. %)
Crosslinking
(mol. %)
7.5 15 7.5 7 3.5
15.0 30 15 30 15
18.8 34 17 34 17
25.0 39 20 44 22
50.0 71 36 76 38
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Figure1
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Figure 2
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Figure 3
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Figure 4
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Figure 5
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Figure 6
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Figure 7
