- Email: [email protected]
Biodegradable chitosan nanogels crosslinked with genipin
Accepted Manuscript Title: “Biodegradable Chitosan Nanogels Crosslinked with Genipin” ´ Authors: Maite Arteche Pujana, Leyre P´erez-Alvarez, Luis Carlos Cesteros Iturbe, Issa Katime PII: DOI: Reference:
S0144-8617(13)00128-8 doi:10.1016/j.carbpol.2013.01.082 CARP 7426
To appear in: Received date: Revised date: Accepted date:
13-11-2012 14-1-2013 15-1-2013
´ Please cite this article as: Pujana, M. A., P´erez-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 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
CARBPOL-D-12-02432-R1.
2
“Biodegradable Chitosan Nanogels Crosslinked with Genipin”
3 Maite Arteche Pujana, Leyre Pérez-Álvarez, Luis Carlos Cesteros Iturbe, Issa
5
Katime.
ip t
4
cr
6
Grupo de Nuevos Materiales y Espectroscopia Supramolecular. Departamento
8
de Química Física. Facultad de Ciencia y Tecnología. Universidad del País
9
Vasco (UPV/EHU). 644 Bilbao, Spain.
11
+34946012711
an
E-mail: [email protected]
M
10
Keywords
14
Genipin, nanogels, chitosan.
te
13
Ac ce p
15
17
d
12
16
us
7
ABSTRACT
Chitosan nanoparticles crosslinked with genipin were prepared by reverse
18
microemulsion that allowed to obtain highly monodisperse (3-20 nm by TEM)
19
nanogels. The incorporation of genipin into chitosan was confirmed and
20
quantitatively evaluated by UV-Vis and 1H NMR. Loosely crosslinked chitosan
21
networks showed higher water solubility at neutral pHs than pure chitosan. The
22
hydrodynamic diameter of the genipin-chitosan nanogels ranged from 270 to
23
390 nm and no remarkable differences were found when the crosslinking degree
24
was varied. The hydrodynamic diameters of the nanoparticles increased slightly
25
at acidic pH and the protonation of ionizable amino groups with the pH was 1 Page 1 of 34
confirmed by the zeta potential measurements. The biocompatible and
27
biodegradable nature, as well as the colloidal and monodisperse particle size of
28
the prepared nanogels, make them attractive candidates for a large variety of
29
biomedical applications.
ip t
26
30 HIGHLIGHTS
cr
31 32
Chitosan was crosslinked with genipin in reverse microemulsion medium.
34
Chitosan-genipin crosslinking reaction was quantitatively evaluated by UV-Vis
35
and 1H NMR.
36
Chitosan-genipin nanogels showed improved water solubility at neutral pHs.
37
Swelling behaviour of the nanogels was studied as function of crosslinking
38
degree and pH.
d
M
an
us
33
te
39 Keywords
41
Genipin, nanogels, chitosan.
42 43 44 45
Ac ce p
40
INTRODUCTION
Chitosan is a linear and partly acetylated (1-4)-2-amino-2-deoxy-β-D-
46
glucan isolated from marine chitin (Muzzarelli, Boudrant, Meyer, Manno,
47
DeMarchis & Paoletti, 2012; Muzzarelli, Jeuniaux, Gooday, 1986). In contrast to
48
other polymers, chitosan is positively charged due to the protonation of its
49
primary amines weak basic groups, which give it special characteristics and
50
feasibility to chemical modification. Chitosan exhibits many favourable
2 Page 2 of 34
characteristics such as biodegradability, nontoxicity and biocompatibility, which
52
give it a great potential in a wide range of areas such as food, cosmetics,
53
wastewater treating, biotechnology, or biomedicine (Shahidi, Arachchi & Jeon,
54
1999; Ravi Kumar, 2004; Crini, 2005)
ip t
51
Chitosan has been extensively studied for delivery of bioactive reagents,
55
including drugs (Felt, Buri & Gurny, 1998), proteins and genes (Malhotra,
57
Kulamarva, Sebak, Paul, Bhathena, Mirzaei & Prakash, 2009) and it is, in this
58
respect, one of the preferred candidates in advanced fields like gene delivery
59
(Liu & Yao, 2002), transnasal delivery (Illum, Farraj & Davis, 1994) or ocular
60
diseases treatment (Alonso & Sánchez, 2003). Regarding this purpose chitosan
61
has been prepared in a wide variety of forms, namely hydrogels, beads, films,
62
tablets, microspheres, composites, micro and nanoparticles (Miyazaki,
63
Nakayama, Oda, Takada & Attwood, 1994; Lee, Nam, Im, Park, Leeb, Seol,
64
Chung & Lee, 2002; Banerjee, Mitra, Kumar Singh, Kumar Sharma & Maitra,
65
2002).
us
an
M
d
te
Nanoparticles have a special role in targeted drug delivery due to their
Ac ce p
66
cr
56
67
long shelf life, easy transport to different body sites and high drug entrapment
68
efficacy (Agnihotri, Mallikarjuna & Aminabhavi, 2004). Chitosan nanoparticles
69
can be obtained by chemical crosslinking leading to quite stable matrixes. These
70
chitosan nanogels are commonly prepared by modification with bifunctional
71
agents such as diisocyanate, diepoxy compounds, dialdehyde and other
72
reagents (Wenling Mingyu, Qiang, Yandao, Nanming & Xiufang, 2005; Rinaudo,
73
2010). Among them, in the last decades glutaraldehyde was the most broadly
74
used crosslinker agent in covalent formulations. However, due to the proved
3 Page 3 of 34
75
toxicity of glutaraldehyde, recently, biocompatible crosslinking agents have
76
received much attention in the field of biomedical application.
77
Extended studies about genipin, an alternative natural crosslinking agent, has shown its ability to form biocompatible and stable crosslinked products
79
10,000 times less cytotoxic than glutaraldehyde (Sung, Huang, Huang & Tsai,
80
1999). Genipin is an iridoid glucoside extracted from Gardenia that has been
81
used in traditional Chinese medicine and as a blue colorant in the food industry.
cr
ip t
78
Genipin reacts under mild conditions with primary amine groups and this
83
reaction, which is undoubtedly identified by mean of its inherent blue coloration,
84
has been employed to crosslink chitosan (Jin, Song & Hourston, 2004) and
85
other amino group containing biomaterials (González, Strumia & Alvarez
86
Igarzabal, 2011).
an
M
Genipin has been widely employed in the field of biomaterials specially in
d
87
us
82
studies of tissue fixation and regeneration in various biomaterials and natural
89
biological tissues, or in studies of drug delivery of macrogels, microspheres and
90
semi-interpenetrating polymer networks (semi-IPNs) (Muzzarelli, Greco,
91
Busilachi, Sollazzo & Gigante, 2012). Additionally, genipin has been employed
92
to obtain nanocomposites consisted of metal nanoparticles embedded in
93
chitosan crosslinked matrixes (Liu & Huang, 2008). However, there are not
94
many publications relating to genipin crosslinked nanoparticles (Choubey &
95
Bajpai, 2010; Maggi, Ciccarelli, Diociaiuti, Casciardi & Masci, 2011) which
96
encourages us the use of genipin as a crosslinking agent to develop chitosan
97
nanogels for biomedical applications.
98 99
Ac ce p
te
88
Since size distribution plays an important role in drug delivery application, it is necessary to prepare uniformly sized nanoparticles. However, the size
4 Page 4 of 34
distribution of chitosan nanoparticles is very difficult to control. Various studies
101
have shown that reverse microemulsion (w/o) is an effective way to prepare low
102
sized particles with uniform particle size distribution. This promising method for
103
the effective preparation of chitosan nanoparticles is a transparent, isotropic and
104
thermodynamically stable synthesis medium. Microemulsions not only act as
105
microreactors for the formation of nanoparticles but also inhibit excessive
106
aggregation of the forming particles (Liu, Shao, Gea & Chena, 2007). Several
107
authors have employed the well stablished microemulsion formulation based on
108
cyclohexane/Triton X100/n-hexane/water for obtaining chitosan nanosized
109
particles (Wang, Wang, Luo & Dai, 2008; Arteche Pujana, Pérez-Alvarez,
110
Cesteros Iturbe & Katime, 2012).
cr
us
an
M
111
ip t
100
In this work chitosan and genipin were chosen for preparing polymeric nanogels by crosslinking of chitosan. Both of these compounds display
113
advantageous biomedical properties. Nanogels were preparing by w/o
114
microemulsion method and their physicochemical properties, such as, particle
115
size, pH-sensitivity swelling, surface charge or water solubility, were studied.
117 118 119 120
te
Ac ce p
116
d
112
EXPERIMENTAL
Materials
Low molecular weight chitosan (Aldrich) was purified using the procedure
121
previously described (Signini & Campana, 1999). The deacetylation degree of
122
chitosan determined by 1H NMR was 79% which is in good agreement with the
123
value reported by the supplier (75-85%). Its viscosity average molecular weight
124
of chitosan measured by an Ubbelohde capillary viscometer (HAc 0.3M/NaAc
5 Page 5 of 34
0.2M, 25ºC) (Rinaudo, Milas & Dung, 1993) was 66,000 g/mol. Genipin, triton X-
126
100 and hexanol (for synthesis, 98%) were purchased from Sigma-Aldrich.
127
Cyclohexane (for synthesis, 98%), acetic acid (for analysis, 99.8%) and ethanol
128
(for analysis, 96%) were supplied from Panreac and D-glucosamine
129
hydrochloride by Acros Organics.
ip t
125
132
Preparation of nanogels
1g of chitosan was dissolved in 100 mL of 1%v/v aqueous acetic acid
us
131
cr
130
solution and separately genipin was dissolved in distilled water (1-540 mg/mL).
134
Chitosan nanoparticles were prepared using a procedure that was
an
133
previously described by Jia et al. (Jia, Yujun & Guangsheng, 2005) with minor
136
modification. Two separate W/O microemulsions were produced. The first
137
microemulsion (MC) was formed by dropwise addition of Triton X-100 to a
138
mixture of cyclohexane: n-hexanol: chitosan solution in ratio of 2.75:1:1
139
respectively. Triton X 100 was added until microemulsion became optically
140
transparent. The second microemulsion (MG) containing genipin solution but
141
without chitosan was prepared with the same procedure. Then, MG
142
microemulsion was added to MC microemulsion under magnetic stirring at
143
different crosslinker stoichiometric ratio. The crosslinking reaction was carried
144
out at 25 ºC for 4 days. During crosslinking reaction the microemulsion became
145
bluish. The microemulsion was broken by adding ethanol to obtain blue colored
146
nanoparticles. Nanogels were washed repeatedly with ethanol by centrifugation
147
(9000rpm, 10 min, at room temperature). Finally prepared nanogels were
148
washed with distilled water, ultrafiltered (OMEGA 76 MM 100K 12/PK) and
149
vacuum dried (55ºC, 24 hours).
Ac ce p
te
d
M
135
6 Page 6 of 34
150 151 152
Methods The nanogels were analyzed with NMR spectroscopy. 1H spectra were obtained on a Bruker Avance 500 MHz instrument and the samples were
154
dissolved in 2% w/w. CD3COOD/ D2O. Solid samples were analyzed by 13C
155
NMR spectroscopy with a Bruker Avance III 400 spectrometer.
cr
ip t
153
A Philips CM120 with Olympus SIS Morada digital camera transmission
157
electron microscope was used to characterize the size and morphology of the
158
dried chitosan nanoparticles. Nanoparticles were prepared dissolving dried
159
powder in 1%v/v aqueous acetic acid solution with a concentration of 1mg/mL.
160
which was then dried in a vacuum chamber.
163
an
M
Vis Spectrophotometer.
d
162
UV–Vis spectroscopy measurements were registered in a Cintra 303 UV-
The genipin-chitosan crosslinking reaction was characterized by UV-Vis.
te
161
us
156
Along the crosslinking reaction, spectra of the microemulsion samples were
165
obtained at different time intervals in a wavelength range from 200 to 700 nm.
166
Furthermore, the quantitative determination of the reacted free amino groups in
167
nanoparticles crosslinked with genipin was carried out by mean of the previous
168
calibration with D-glucosamine in the microemulsion medium. D-glucosamine
169
(50 to 750 mg/L) and genipin (500 mg/L) after being dissolved in the described
170
microemulsion medium were incubated at 25ºC. The absorbance at 605 nm was
171
measured and the obtained linear regression equation was A = 0.0188+
172
0.0021X (R2 = 0.99764).
Ac ce p
164
173
UV-Vis spectroscopy was also employed for the study of the water
174
solubility of the nanogels at different pH values by mean of the measurement of
7 Page 7 of 34
the transmittance of the nanogels dispersions at 750 nm. Nanogels were
176
dissolved in 1% HAc solution (1 mg/5 mL), and the transmittance was measured
177
24 hours after the pH of the solution was adjusted by the addition of 2 N NaOH
178
solution.
179
ip t
175
Dynamic laser light scattering measurements for determining the size of
the nanogels were performed using a Brookhaven BI-9000AT with a goniometer.
181
A water-cooled argon-ion laser was operated at 514.5 nm as the light source.
182
The time dependence of the intensity autocorrelation function of the scattered
183
intensity was obtained by using a 522 channel digital correlator. The size of the
184
nanoparticles was determined from the diffusion intensity of the particles using
185
the Stokes-Einstein equation. The size distribution was obtained by NNLS
186
analysis and the uncertainties of the measurements represented the standard
187
deviation of the mean of the replicate runs. The dried powder samples were
188
dispersed in 1% HAc solution (1mg/mL) during 3 days and then diluted until the
189
concentration was 40 mg/L. All measurements were made at room temperature.
190
The pH was changed with 2 N NaOH solution.
us
an
M
d
te
Ac ce p
191
cr
180
X-ray diffraction patterns were obtained by a Transmision STOE Stadip
192
X-ray diffractometer with a goniometer speed of 0.1º/90s. The range of
193
diffraction angle 2 was 2.5-34º.
194
Electrophoretic mobility measurements were performed with a Zeta-Sizer
195
IV (Malvern Instruments). Nanogels were dispersed in 1% HAc solution
196
(1mg/mL) during 3 days and finally diluted to a concentration of 40 mg/L. Data
197
were obtained from the average of at least ten measurements in universal cells.
198
The external pH was varied by adding 2 N NaOH solution.
199
8 Page 8 of 34
200
RESULTS AND DISCUSSION
201 202 1.- Study of the crosslinking reaction
ip t
203 204
Covalently crosslinked chitosan nanoparticles were prepared within a
cr
205
reverse microemulsion medium in order to control the colloidal particle size of
207
the prepared nanogels. This modification reaction took place when separately
208
prepared chitosan and genipin microemulsion were mixed as described above.
209
This crosslinking occurs by mean of a complex mechanism which is believed
210
that consists in two reactions involving different sites on the genipin molecule
211
(Butler, Ng & Pudney, 2003) and produces dark-blue coloration in the reaction
212
mixture. The appearance of this coloration in the reaction mixture of genipin and
213
chitosan is traditionally considered as rapid evidence that the crosslinking
214
reaction is taking place (Butler, Ng & Pudney, 2003; Muzzarelli, 2009).
an
M
d
te
The evolution of the crosslinking reaction was monitored by mean of the
Ac ce p
215
us
206
216
UV-Vis spectra of the final microemulsion. As can be seen in Figure 1A, a new
217
peak appeared at 605 nm, which corresponds to the formed blue pigment, and
218
continued to increase in intensity throughout the course of the reaction.
219
Ramos et al. (Ramos-Ponce, Vega, Sandoval-Fabián, Colunga-Urbina,
220
Balagurusamy, Rodriguez-Gonzalez & Contreras-Esquivel, 2010) described an
221
efficient colorimetric method using genipin as a specific reagent for quantitative
222
determination of free amino groups of chitin derivatives. Following this
223
procedure, the relationship between the absorbance of the microemulsion at 605
224
nm and the concentration of free amino groups in the nanogels was determined
9 Page 9 of 34
by mean of a calibration curve with standard solutions of D-glucosamine. The
226
method reported by Ramos et al. could be applied to the microemulsion medium
227
after slight modifications. The concentration of modified free amino groups as a
228
function of reaction time was analyzed in order to optimize the reaction time.
229
Results of this study are shown in Figure 1B, demonstrating that after 7 days the
230
end point of the reaction was reached.
cr
ip t
225
231 3.- NMR characterization
us
232 233
The success of the modification reaction was also confirmed and
an
234
quantitatively evaluated by 1H NMR. Pure chitosan shows a peak at 2.0–2.1
236
ppm corresponding to the three protons of N-acetyl glucosamine (GlcNAc) and a
237
peak at 3.1–3.2 ppm due to H-2 proton of glucosamine (GlcN) residues which
238
represent the primary amino group content of the chitosan. Figure 2 displays the
239
spectra of several genipin-chitosan nanogels. A clear decrease of the signal at
240
3.2 ppm was observed for all the samples, indicating the loss of amine groups
241
by reaction with genipin showing that the addition of genipin to chitosan was
242
successfully carried out.
d
te
Ac ce p
243
M
235
The peak of the 1H NMR spectrum of chitosan corresponding to the
244
methyl protons at 2.0 - 2.1 ppm possess the highest resolution (Kasaai, 2010)
245
and was used for the determination of the free amino content by mean of the
246
integration of the resonance of H-2 protons at 3.2 ppm. Thus, the modification
247
degree of the chitosan chains was evaluated by the decrease of the
248
glucosamine signal (3.2 ppm). The crosslinking grade was calculated assuming
249
that the reaction occurs on both reaction sites of the genipin molecule. Table 1
10 Page 10 of 34
lists the nanogels compositions obtained by 1H NMR analysis for each initial
251
reaction feed. The data collected in Table 1 show a rather good correlation
252
between genipin stoichiometric ratio (with respect to the total amount of
253
glucosamine units) in initial reaction mixture and final modification degree in the
254
nanogels, especially for intermediate compositions. These results suggest that
255
the linkage genipin-chitosan occurs in a ratio around 1:2 and therefore the self-
256
polymerization of genipin is not likely to take place. However, for high crosslinker
257
content, the ratio genipin feed: modified chitosan increased. Assuming that the
258
reaction yields did not varied significantly, it could be related to a higher
259
probability that polymerization takes place. This in accordance with an
260
intermediate brownish colour appeared during crosslinking reaction of these
261
samples before the blue intense coloration (Mi, Shyu & Peng, 2005).
cr
us
an
M
262
ip t
250
On another hand, modification degrees of chitosan in the nanogels obtained by 1H NMR analysis agreed with those obtained by UV-Vis
264
spectroscopy (Table 1).
te
Besides, 13C NMR spectroscopy also corroborates the modification of
Ac ce p
265
d
263
266
linear chitosan with genipin. Figure 3 shows the 13C NMR of initial pure chitosan
267
and chitosan crosslinked with genipin. The crosslinking of chitosan with genipin
268
results in some slight changes on the 13 C NMR spectrum of chitosan. The
269
resonance signal of chitosan C-4 at 81-87 ppm decreased and becomes into a
270
shoulder. Mi et al. (Mi, Sung & Shyu, (2000); Mi, Sung & Shyu, 2001) attributed
271
this fact to a conformational change from the linear structure of original chitosan
272
to crosslinked chitosan, revealing the formation of a secondary amide and
273
heterocyclic amino linkage, and so, the crosslinking of chitosan. Additionally, a
274
slight shift of C-1 resonance from 112 ppm to a broad resonance centered at
11 Page 11 of 34
275
around 109 ppm occurs, which is attributed to the incorporation of the
276
heterocyclic ring at C-2 (Mi, Sung & Shyu, 2000).
277
279
4.- Water solubility
ip t
278
The applications of chitosan nanoparticles suffer severe limitations
because they are no dispersible in neutral or alkaline pH aqueous media. This
281
water insolubility in high pH solutions is due to the presence of rigid crystalline
282
domains, formed by intra- and/or intermolecular hydrogen bonding (Nishimura,
283
Kohgo, Kurita & Kuzuhar, 1991).
us
The solubility of the prepared genipin-chitosan nanogels was
an
284
cr
280
characterized as a function of pH and compared with the unmodified chitosan.
286
Figure 4A shows the transmittance at 750 nm of aqueous solutions of linear
287
chitosan and genipin-chitosan nanogels prepared in the above described
288
conditions.
d te
289
M
285
In general, as the pH decreases the protonation of free amino groups of chitosan chains takes place and the H-bonding interactions decreases,
291
improving water-polymer interaction and consequently increasing the
292
transmittance of aqueous dispersions (Figure 4A). So, when pH increases the
293
dispersions become opaque and particles aggregation takes places.
294
Ac ce p
290
The solubility of the nanogels was determined from pH50 parameter that is
295
defined as the pH value when the transmittance at 750 nm reached 50%.
296
Loosely crosslinked nanogels (< 7.5 mol. % genipin) showed a slightly improved
297
stability with the pH, displaying a value of 7.3 for the pH50 parameter, while the
298
value measured for pure chitosan was 7.0. When the genipin content was higher
12 Page 12 of 34
299
than 7.5 mol. % the pH50 of the nanogels was lower than that of the pure
300
chitosan in all the cases.
301
It is well kwon that the modification of chitosan with more or less bulky pendant groups leads to the reduction of hydrogen bonding in
303
chitosan and therefore, the solubility of the chitosan derivatives increases. In this
304
regard, the increase in water solubility of PEGylated chitosan (Chan, Kurisawa,
305
Chung & Yang, 2007) has been attributed to the decrease of intermolecular
306
interactions, as well as the swelling of N-alkyl and N-acylated chitosans in
307
water, in spite of their hydrophobicity (Ravi Kumar, 2000; Shasiwa, Kawasaki,
308
Nakayama, Muraki, Yamamoto & Aiba, 2002).
cr
us
an
In general, chitosan derivatives that precipitate in basic medium have a
M
309
ip t
302
high degree of crystallinity. On the contrary, chitosan obtained with amorphous
311
form and loosely aggregated state is soluble in water (Lu, Song, Cao, Chen &
312
Yao, 2004).
te
d
310
Figure 4B shows the X-ray diffractograms of some of the samples whose
314
solubility behaviour is displayed in Figure 4A. Pure chitosan sample displays the
315
typical pattern of chitosan substrates with peaks at 2 around 11 and 20º. As
316
can be observed, the chemical crosslinking with genipin causes the loss of
317
crystallinity in the samples. All the studied genipin-chitosan nanogels displayed
318
a difractogram of an amorphous material.
319
Ac ce p
313
Thus, it could be concluded that a moderate crosslinking disturbs the intra
320
and inter intermolecular hydrogen bonding resulting in the observed
321
enhancement in solubility. However, as crosslinking increases (up to 7.5 mol.
322
%), in spite of the weakened intra-intermolecular interactions of chitosan chains,
13 Page 13 of 34
323
the nanogels become more and more compact and a close and rigid network is
324
generated, leading to a substantial reduction of the water solubility.
325 5.- Particle size and swelling properties of the nanogels
ip t
326 327
The morphology and particle size distribution of dried nanogels was
cr
328
studied by TEM. TEM images (Figure 5A) indicate that genipin-chitosan
330
nanogels are spherical, have nearly uniform particle size distribution (Figure 5B)
331
and there is no severe particle agglomeration, which are very interesting
332
qualities for biomedical applications, such as drug delivery. The average
333
diameters of nanoparticles ranged from 3 to 20 nm.
M
an
us
329
The particle size of swollen nanogels was determined by QELS
335
measurements. Due to the adhesive nature of chitosan in aqueous solution,
336
these nanoparticles tend to form aggregates that lead to a higher average
337
hydrodynamic diameter than the actual size of the particles (Wu, Zhou & Wang,
338
1995). The progressive dilution of the nanoparticle dispersions has been
339
reported as an effective way to reduce the inter-particle interaction and the
340
presence of large aggregates of chitosan is (Sorlier, Rochas, Morfin, Viton &
341
Domard, 2003) and was applied in this study. However, the solubility study
342
revealed in comparison with pure chitosan a higher tendency of genipin-chitosan
343
nanogels to aggregate, except for loosely crosslinked ones (7.5 mol. %).
344
Ac ce p
te
d
334
Figure 6 shows the average diameters of the nanogels determined by
345
QELS at pH = 4.0 for several genipin stoichiometric ratios. No strong correlation
346
was found between the hydrodynamic diameter of the genipin-chitosan nanogels
347
and the crosslinking degree. Besides, as is shown in Figure 7, a slight pH-
14 Page 14 of 34
348
responsive behaviour was observed. Thus, it could be concluded that the
349
nanogels display a weak swelling capacity due to the rigid and hydrophobic
350
nature of the employed crosslinker. The swelling effect induced in an ionic network, such as chitosan
352
nanogels, can be considered as the accumulative effect of the mixing, elastic
353
and ionic contributions (Flory, & Rehner, 1943). In the case of the genipin-
354
chitosan nanogels at pH = 4, an increase in crosslinking has opposite effects.
355
On the one hand leads to the diminishing of ionic contribution due to the
356
decrease of ionizable groups in the network, and to the enhancement of the
357
elastic force. Both of these effects tend to limit the swelling of the gel. However,
358
as crosslinker concentration increases (up to 7.5 mol. %), the mixing
359
contribution is drastically reduced, due to the hydrophobic and closed nature of
360
the network, and aggregation takes place, increasing the measured
361
hydrodynamic diameter. This may explain that the determined particle sizes of
362
the nanoparticles varying the content of genipin were fairly similar each other
363
(Figure 6).
cr
us
an
M
d
te
Ac ce p
364
ip t
351
At constant degree of crosslinking, when the external pH was varied it
365
was found that the nanoparticles swell slightly at pH below 5.5 (Figure 7). This
366
may be due to the protonation of free amine groups of chitosan chains at low
367
pH, which results in an electrostatic repulsion charge favouring the penetration
368
of water into the gel. For basic pHs the precipitation of the nanogels takes place
369
and particle size or could not be measured or increased as consequences of
370
particle aggregation. Studies of the pH-responsive swelling of genipin-chitosan
371
macrogels also did not displayed an abrupt phase transition (Kaminski, K.,
372
Zazakowny, K., Szczubiałka, K., Nowakowska M., 2008) that was observed in
15 Page 15 of 34
other studied chitosan networks (Arteche Pujana, Pérez-Alvarez, Cesteros
374
Iturbe & Katime, 2012). In this sense, Jharen et al. (Jahren, Butler, Adams &
375
Cameron, 2010) found that the pH-responsive phase transition decreased with
376
crosslinking in genipin-chitosan hydrogels and disappeared completely for a
377
genipin percentage of 80 mol. %.
To obtain information about the electrical state of the ionizable groups
cr
378
ip t
373
responsible of the pH-responsive behaviour of the nanoparticles, zeta potential
380
of genipin-chitosan nanogels was measured as function of genipin content
381
(Figure 6), as well as, of pH (Figure 7). In all the samples the value of the zeta
382
potential increased up to 20 mV when the pH increased from 4 to 9. All the
383
nanogels showed positive charge at acidic conditions, so all of them maintained
384
the chitosan mucoadhesive and absorption enhancement potential properties.
385
It is shown in Figure 6 that the zeta potential values of protonated
386
networks (pH = 4.0) decreased as the nanogels crosslinking was higher, proving
387
the loss of free amine moiety along the chitosan backbone within the
388
modification with genipin. Regarding pH (Figure 7), as the chitosan chains
389
contain free amine pH-ionizable groups, a pH variation modifies the electrical
390
state of the network. The amine primaries groups are protonated in acidic media,
391
and the surface charge is positive and increases along the ionization process
392
occurs. So, the progressive protonation of amino groups as pH decreases was
393
confirmed by measuring the electrokinetic potential of the nanogels.
Ac ce p
te
d
M
an
us
379
394 395
CONCLUSIONS
396
16 Page 16 of 34
397
Chitosan low-size nanogels were prepared by the combination of two well-known methods such as, reverse microemulsion and crosslinking by
399
genipin. Nanogels sizes ranging from 3-20 nm determined by TEM, and 270-390
400
nm determined by QELS in aqueous media, could be obtained by this covalent
401
crosslinking method. Loosely crosslinked nanogels displayed an improved water
402
solubility compared with pure chitosan. The variation of water solubility of
403
chitosan due to the crosslinking with genipin could be consider as a compromise
404
between the decrease of crystallinity and the elastic force within the generated
405
network. This elastic contribution may play an essential role in the water
406
solubility and pH-sensitive swelling of the nanogels due to the low molecular
407
weight and rigid nature of genipin molecule.
M
an
us
cr
ip t
398
408 409
ACKNOWLEDGEMENTS
413 414 415 416 417
te
412
Financial support for this work was provided by the Basque Government and the University of the Basque Country (UPV/EHU), for awarding a fellowship.
Ac ce p
411
d
410
REFERENCES
Agnihotri, S. A., Mallikarjuna, N. N. & Aminabhavi, T. M. (2004). Recent
418
advances on chitosan-based micro- and nanoparticles in drug delivery.
419
Journal of Controlled Release, 100, 5–28.
420 421
Alonso, M. J. & Sánchez, A. (2003). The potential of chitosan in ocular drug delivery. Journal of Pharmacy and Pharmacology, 55, 1451–1463.
17 Page 17 of 34
422
Arteche Pujana, M., Pérez-Alvarez, L., Cesteros Iturbe, L. C. & Katime, I.
423
(2012). Water dispersible pH-responsive chitosan nanogels modified with
424
biocompatible crosslinking-agents. Polymer, 53, 3107-3116. Banerjee, T., Mitra, S., Kumar Singh, A., Kumar Sharma, R. & Maitra, A. (2002).
ip t
425
Preparation, characterization and biodistribution of ultrafine chitosan
427
nanoparticles. International Journal of Pharmaceutics, 243, 93–105.
428
cr
426
Butler, M.F., Ng, Y.F. & Pudney, P.D.A. (2003). Mechanism and kinetics of the crosslinking reaction between biopolymers containing primary amine groups
430
and genipin. Journal of Polymer Science. Part A: Polymer Chemistry, 41,
431
3941–3953
an
Chan, P., Kurisawa, M., Chung, J. E & Yang, Y. Y. (2007). Synthesis and
M
432
us
429
characterization of chitosan-g-poly(ethylene glycol)-folate as a non-viral
434
carrier for tumor-targeted gene delivery. Biomaterials, 28, 540-549.
d
433
Choubey, J. & Bajpai, A. K. (2010). Investigation on magnetically controlled
436
delivery of doxorubicin from superparamagnetic nanocarriers of gelatin
437
crosslinked with genipin. Journal of Materials Science- Materials in Medicine,
439 440 441 442 443 444 445
Ac ce p
438
te
435
21, 1573–1586.
Crini, G. (2005). Recent developments in polysaccharide-based materials used as adsorbents in wastewater treatment. Progress in Polymer Science 30, 38– 70.
Felt, O., Buri, P. & Gurny, R. (1998). Chitosan: a unique polysaccharide for drug delivery. Drug Development and Industrial Pharmacy, 24, 979– 993. Flory, P. J. & Rehner, J. (1943). Statistical Mechanics of Cross-Linked Polymer Networks II. Swelling. Journal of Chemical Physics, 11, 521.
18 Page 18 of 34
446
González, A., Strumia, M. C. & Alvarez Igarzabal, C. I. (2011). Cross-linked soy
447
protein as material for biodegradable films: synthesis, characterization and
448
biodegradation. Journal of Food Engineering, 106, 331–338
450
Illum, L., Farraj, N.F. & Davis, S.S. (1994). Chitosan as a novel nasal delivery
ip t
449
system for peptide drugs. Pharmaceutical Research, 11, 1186– 1189.
Jahren, S. L., Butler, M. F., Adams, S. & Cameron, R. E. (2010). Swelling and
452
Viscoelastic Characterisation of pH-Responsive Chitosan Hydrogels for
453
Targeted Drug Delivery. Macromolecular Chemistry and Physics, 211, 644–
454
650.
us
Jia, Z., Yujun, W. & Guangsheng L. (2005). Adsorption of diuretic furosemide
an
455
cr
451
onto chitosan nanoparticles prepared with a water-in-oil nanoemulsion
457
system. Reactive & Functional Polymers, 65, 249–257. Jin, J., Song, M. & Hourston, D. J. (2004). Novel chitosanbased films cross-
d
458
M
456
linked by genipin with improved physical properties. Biomacromolecules, 5,
460
162-168.
te
459
Kaminski, K., Zazakowny, K., Szczubiałka, K. & Nowakowska M. (2008). pH-
462
sensitive genipin-cross-linked chitosan microspheres for heparin removal.
463
Ac ce p
461
Biomacromolecules, 9, 3127–3132.
464
Kasaai, M. R. (2010). Determination of the degree of N-acetylation for chitin and
465
chitosan by various NMR spectroscopy techniques: A review. Carbohydrate
466
Polymers, 79, 801–810.
467
Lee, J.-Y., Nam, S.-H., Im, S.-Y., Park, Y.-J., Leeb, Y.-M., Seol Y.-J., Chung, C.-
468
P. & Lee S.-J. (2002). Enhanced bone formation by controlled growth factor
469
delivery from chitosan-based biomaterials. Journal of Controlled Release, 78,
470
187–197.
19 Page 19 of 34
471
Liu, B. S. & Huang, T. B. (2008). Nanocomposites of Genipin-Crosslinked
472
Chitosan/Silver Nanoparticles–Structural Reinforcement and Antimicrobial
473
Properties. Macromolecular Bioscience, 8, 932–941.
476
ip t
475
Liu, G., Shao, L., Gea, F. & Chena, J. (2007). Preparation of ultrafine chitosan particles by reverse microemulsion. China Particuology, 5, 384–390.
Liu, W.G. & Yao, K.D. (2002). Chitosan and its derivatives--a promising non-viral
cr
474
vector for gene transfection. Journal of Controlled Release, 83, 1– 11.
478
Lu, S., Song, X., Cao, D., Chen, Y. & Yao K. (2004). Preparation of Water-
479
us
477
Soluble Chitosan. Journal of Applied Polymer Science, 91, 3497–3503. Maggi, F., Ciccarelli, S., Diociaiuti, M., Casciardi, S. & Masci G. (2011). Chitosan
481
Nanogels by Template Chemical Cross-Linking in Polyion Complex Micelle
482
Nanoreactors. Biomacromolecules, 12, 3499−3507
M
an
480
Malhotra, M., Kulamarva, A., Sebak, S., Paul, A., Bhathena, J., Mirzaei M. &
484
Prakash, S. (2009). Ultrafine chitosan nanoparticles as an efficient nucleic
485
acid delivery system targeting neuronal cells. Drug Development and
486
Industrial Pharmacy 35, 719–726.
Ac ce p
te
d
483
487
Mi, F.-L., Sung, H.-W. & Shyu, S.-S. (2000). Synthesis and characterization of a
488
novel chitosan-based network prepared using naturally occurring crosslinker.
489 490 491
Journal of Polymer Science- Part A: Polymer Chemistry, 38, 2804–2814.
Mi, F.-L. Sung, H.-W. & Shyu; S.-S. (2001). Release of Indomethacin from a Novel Chitosan Microsphere Prepared by a Naturally Occurring Crosslinker:
492
Examination of Crosslinking and Polycation–Anionic Drug Interaction. Journal
493
of Applied Polymer Science, 81, 1700–1711.
494 495
Mi, F.-L., Shyu, S.-S. & Peng, C.-K. (2005). Characterization of ring-opening polymerization of genipin and pH-dependent cross-linking reactions between
20 Page 20 of 34
496
chitosan and genipin. Journal of Polymer Science- Part A: Polymer
497
Chemistry, 43, 1985–2000.
498
Miyazaki, S. Nakayama A, Oda M, Takada M & Attwood D. (1994). Chitosan and sodium alginate based bioadhesive tablets for intraoral drug delivery.
500
Biological & Pharmaceutical Bulletin, 17, 745-7.
503 504
cr
502
Muzzarelli, R.A.A., Jeuniaux, C. & Gooday, G. W. (1986). Chitin in nature and Technology. New York: Plenum Press.
Muzzarelli, RAA. (2009). Genipin-crosslinked chitosan hydrogels as biomedical
us
501
ip t
499
and pharmaceutical aids. Carbohydrate Polymers, 77, 1-9.
Muzzarelli, R.A.A., Boudrant, J., Meyer, D., Manno, N., DeMarchis, M. &
506
Paoletti, M.G. (2012). Current views on fungal chitin/chitosan, human
507
chitinases, food preservation, glucans, pectins and inulin: A tribute to Henri
508
Braconnot, precursor of the carbohydrate polymers science, on the chitin
509
bicentennial. Carbohydrate Polymers, 87, 995-1012.
512 513 514 515 516 517
M
d
te
511
Muzzarelli, R.A.A., Greco, F., Busilachi, A., Sollazzo, V. & Gigante, A. (2012). Chitosan, hyaluronan and chondroitin sulfate in tissue engineering for
Ac ce p
510
an
505
cartilage regeneration: a review. Carbohydrate polymers, 89, 723-739.
Nishimura, S.I., Kohgo, O., Kurita, K. & Kuzuhara, H. (1991). Chemospecific manipulations of a rigid polysaccharide: syntheses of novel chitosan derivatives with excellent solubility in common organic solvents by regioselective chemical modifications. Macromolecules, 24, 4745–4748.
Ramos-Ponce, L. M, Vega, M., Sandoval-Fabián, G. C., Colunga-Urbina, E.,
518
Balagurusamy, N., Rodriguez-Gonzalez, F. J. & Contreras-Esquivel, J, C.
519
(2010). A Simple Colorimetric Determination of the Free Amino Groups in
21 Page 21 of 34
520
Water Soluble Chitin Derivatives Using Genipin. Food Science &
521
Biotechnology, 19, 683-689.
524 525 526
Reactive & Functional Polymers, 46, 1–27.
ip t
523
Ravi Kumar, M.N.V. (2000). A review of chitin and chitosan applications.
Ravi Kumar, M.N.V. (2004). Chitosan chemistry and pharmaceutical perspectives. Chemical Reviews, 104, 6017–6084.
cr
522
Rinaudo, M., Milas, M. & Dung, P.L. Characterization of chitosan. Influence of ionic strength and degree of acetylation on chain expansion. (1993).
528
International Journal of Biological Macromolecules, 15, 281-285.
531 532
an
European Polymer Journal, 46, 1537-1544.
M
530
Rinaudo, M. (2010). New way to crosslink chitosan in aqueous solution.
Shahidi, F., Arachchi, J. K. V. & Jeon, Y.-J. (1999). Food applications of chitin and chitosans. Trends in Food Science & Technology, 10, 37-51.
d
529
us
527
Shasiwa, H., Kawasaki, N., Nakayama, A., Muraki, E., Yamamoto, N. & Aiba, S.
534
(2002). Chemical modification of Chitosan. 14: Synthesis of water-soluble
535
chitosan derivatives by simple acetylation. Biomacromolecules, 3, 1126-1128.
537 538 539 540 541
Ac ce p
536
te
533
Signini, R. & Campana Filho S. P. (1999). On the preparation and characterization of chitosan hydrochloride. Polymer Bulletin, 42, 159–166.
Sung , H. W., Huang, R. N., Huang, L. L. & Tsai, L. L. (1999). In vitro evaluation of cytotoxicity of a naturally occurring cross-linking reagent for biological tissue fixation. Journal of Biomaterials Science, Polymer Edition, 10, 63-78.
Sorlier, P., Rochas, C, Morfin, I., Viton, C. & Domard, A. (2003). Light Scattering
542
Studies of the Solution Properties of Chitosans of Varying Degrees of
543
acetylation. Biomacromolecules, 4, 1034-1040.
22 Page 22 of 34
544
Wang, Y., Wang, X., Luo, G. & Dai, Y. (2008). Adsorption of bovin serum
545
albumin (BSA) onto the magnetic chitosan nanoparticles prepared by a
546
microemulsion system. Bioresource Technology, 99, 3881–3884. Wenling, C., Mingyu, C., Qiang, A., Yandao, G., Nanming, Z. & Xiufang, Z.
ip t
547
(2005). Physical, mechanical and degradation properties, and Schwann cell
549
affinity of cross-linked chitosan films. Journal of Biomaterials Science,
550
Polymer Edition, 16, 791-807.
us
552
Wu, C., Zhou, S. & Wang, W. (1995). A dynamic laser light-scattering study of chitosan in aqueous solution. Biopolymers, 35, 385-392.
an
551
cr
548
553
M
554 555
559 560 561 562 563 564
te
558
Ac ce p
557
d
556
565 566
23 Page 23 of 34
FIGURE CAPTIONS
FIGURE 1. Evolution of A) the visible absorption spectrum of chitosan
ip t
microemulsion during crosslinking reaction with genipin and B) modification degree of chitosan with crosslinking reaction time for: ○) 7.5, □) 12.5, ●) 18.8, ■)
cr
37.5 mol. % of genipin.
us
FIGURE 2. 1H NMR spectrum of genipin-chitosan nanogels (7.5 mol. % of genipin) and amplified resonance signal corresponding to the Ha protons
an
(chitosan H-2 of GlcN ) of nanogels crosslinked with different genipin feeds (0,
M
7.5, 15 and 50 mol. %).
d
FIGURE 3. 13 C NMR spectrum of (A) chitosan nanoparticles crosslinked with
te
genipin and (B) pure chitosan.
Ac ce p
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.
24 Page 24 of 34
FIGURE 6. (□) Zeta potential (●) and Particle size determined by QELS for genipin-chitosan nanogels at pH = 4.0 as function of crosslinker content.
ip t
FIGURE 7. (●) Average hydrodynamic diameter and (○) zeta potential of
Ac ce p
te
d
M
an
us
cr
nanogels as function of external pH (for 12.5 mol. % crosslinker).
25 Page 25 of 34
HIGHLIGHTS
Chitosan was crosslinked with genipin in reverse microemulsion medium.
ip t
Chitosan-genipin crosslinking reaction was quantitatively evaluated by UV-Vis and 1H NMR.
cr
Chitosan-genipin nanogels showed improved water solubility at neutral pHs.
Swelling behaviour of the nanogels was studied as function of crosslinking
Ac ce p
te
d
M
an
us
degree and pH.
26 Page 26 of 34
TABLES
TABLE 1. Modification degree of obtained genipin-chitosan nanogels analyzed by 1H
Genipin Feed
UV-Vis
cr
H NMR
Modification Crosslinking Modification Crosslinking
us
1
ip t
NMR and UV-Vis spectroscopy.
(stoichiometric
degree
mol.%)
(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
(mol. %)
degree
(mol. %)
Ac ce p
te
d
M
an
(mol. %)
27 Page 27 of 34
Ac
ce
pt
ed
M
an
us
cr
i
Figure1
Page 28 of 34
Ac
ce
pt
ed
M
an
us
cr
i
Figure 2
Page 29 of 34
Ac
ce
pt
ed
M
an
us
cr
i
Figure 3
Page 30 of 34
Ac ce p
te
d
M
an
us
cr
ip t
Figure 4
Page 31 of 34
Ac
ce
pt
ed
M
an
us
cr
i
Figure 5
Page 32 of 34
Ac
ce
pt
ed
M
an
us
cr
i
Figure 6
Page 33 of 34
Ac
ce
pt
ed
M
an
us
cr
i
Figure 7
Page 34 of 34
S0144-8617(13)00128-8 doi:10.1016/j.carbpol.2013.01.082 CARP 7426
To appear in: Received date: Revised date: Accepted date:
13-11-2012 14-1-2013 15-1-2013
´ Please cite this article as: Pujana, M. A., P´erez-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 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
CARBPOL-D-12-02432-R1.
2
“Biodegradable Chitosan Nanogels Crosslinked with Genipin”
3 Maite Arteche Pujana, Leyre Pérez-Álvarez, Luis Carlos Cesteros Iturbe, Issa
5
Katime.
ip t
4
cr
6
Grupo de Nuevos Materiales y Espectroscopia Supramolecular. Departamento
8
de Química Física. Facultad de Ciencia y Tecnología. Universidad del País
9
Vasco (UPV/EHU). 644 Bilbao, Spain.
11
+34946012711
an
E-mail: [email protected]
M
10
Keywords
14
Genipin, nanogels, chitosan.
te
13
Ac ce p
15
17
d
12
16
us
7
ABSTRACT
Chitosan nanoparticles crosslinked with genipin were prepared by reverse
18
microemulsion that allowed to obtain highly monodisperse (3-20 nm by TEM)
19
nanogels. The incorporation of genipin into chitosan was confirmed and
20
quantitatively evaluated by UV-Vis and 1H NMR. Loosely crosslinked chitosan
21
networks showed higher water solubility at neutral pHs than pure chitosan. The
22
hydrodynamic diameter of the genipin-chitosan nanogels ranged from 270 to
23
390 nm and no remarkable differences were found when the crosslinking degree
24
was varied. The hydrodynamic diameters of the nanoparticles increased slightly
25
at acidic pH and the protonation of ionizable amino groups with the pH was 1 Page 1 of 34
confirmed by the zeta potential measurements. The biocompatible and
27
biodegradable nature, as well as the colloidal and monodisperse particle size of
28
the prepared nanogels, make them attractive candidates for a large variety of
29
biomedical applications.
ip t
26
30 HIGHLIGHTS
cr
31 32
Chitosan was crosslinked with genipin in reverse microemulsion medium.
34
Chitosan-genipin crosslinking reaction was quantitatively evaluated by UV-Vis
35
and 1H NMR.
36
Chitosan-genipin nanogels showed improved water solubility at neutral pHs.
37
Swelling behaviour of the nanogels was studied as function of crosslinking
38
degree and pH.
d
M
an
us
33
te
39 Keywords
41
Genipin, nanogels, chitosan.
42 43 44 45
Ac ce p
40
INTRODUCTION
Chitosan is a linear and partly acetylated (1-4)-2-amino-2-deoxy-β-D-
46
glucan isolated from marine chitin (Muzzarelli, Boudrant, Meyer, Manno,
47
DeMarchis & Paoletti, 2012; Muzzarelli, Jeuniaux, Gooday, 1986). In contrast to
48
other polymers, chitosan is positively charged due to the protonation of its
49
primary amines weak basic groups, which give it special characteristics and
50
feasibility to chemical modification. Chitosan exhibits many favourable
2 Page 2 of 34
characteristics such as biodegradability, nontoxicity and biocompatibility, which
52
give it a great potential in a wide range of areas such as food, cosmetics,
53
wastewater treating, biotechnology, or biomedicine (Shahidi, Arachchi & Jeon,
54
1999; Ravi Kumar, 2004; Crini, 2005)
ip t
51
Chitosan has been extensively studied for delivery of bioactive reagents,
55
including drugs (Felt, Buri & Gurny, 1998), proteins and genes (Malhotra,
57
Kulamarva, Sebak, Paul, Bhathena, Mirzaei & Prakash, 2009) and it is, in this
58
respect, one of the preferred candidates in advanced fields like gene delivery
59
(Liu & Yao, 2002), transnasal delivery (Illum, Farraj & Davis, 1994) or ocular
60
diseases treatment (Alonso & Sánchez, 2003). Regarding this purpose chitosan
61
has been prepared in a wide variety of forms, namely hydrogels, beads, films,
62
tablets, microspheres, composites, micro and nanoparticles (Miyazaki,
63
Nakayama, Oda, Takada & Attwood, 1994; Lee, Nam, Im, Park, Leeb, Seol,
64
Chung & Lee, 2002; Banerjee, Mitra, Kumar Singh, Kumar Sharma & Maitra,
65
2002).
us
an
M
d
te
Nanoparticles have a special role in targeted drug delivery due to their
Ac ce p
66
cr
56
67
long shelf life, easy transport to different body sites and high drug entrapment
68
efficacy (Agnihotri, Mallikarjuna & Aminabhavi, 2004). Chitosan nanoparticles
69
can be obtained by chemical crosslinking leading to quite stable matrixes. These
70
chitosan nanogels are commonly prepared by modification with bifunctional
71
agents such as diisocyanate, diepoxy compounds, dialdehyde and other
72
reagents (Wenling Mingyu, Qiang, Yandao, Nanming & Xiufang, 2005; Rinaudo,
73
2010). Among them, in the last decades glutaraldehyde was the most broadly
74
used crosslinker agent in covalent formulations. However, due to the proved
3 Page 3 of 34
75
toxicity of glutaraldehyde, recently, biocompatible crosslinking agents have
76
received much attention in the field of biomedical application.
77
Extended studies about genipin, an alternative natural crosslinking agent, has shown its ability to form biocompatible and stable crosslinked products
79
10,000 times less cytotoxic than glutaraldehyde (Sung, Huang, Huang & Tsai,
80
1999). Genipin is an iridoid glucoside extracted from Gardenia that has been
81
used in traditional Chinese medicine and as a blue colorant in the food industry.
cr
ip t
78
Genipin reacts under mild conditions with primary amine groups and this
83
reaction, which is undoubtedly identified by mean of its inherent blue coloration,
84
has been employed to crosslink chitosan (Jin, Song & Hourston, 2004) and
85
other amino group containing biomaterials (González, Strumia & Alvarez
86
Igarzabal, 2011).
an
M
Genipin has been widely employed in the field of biomaterials specially in
d
87
us
82
studies of tissue fixation and regeneration in various biomaterials and natural
89
biological tissues, or in studies of drug delivery of macrogels, microspheres and
90
semi-interpenetrating polymer networks (semi-IPNs) (Muzzarelli, Greco,
91
Busilachi, Sollazzo & Gigante, 2012). Additionally, genipin has been employed
92
to obtain nanocomposites consisted of metal nanoparticles embedded in
93
chitosan crosslinked matrixes (Liu & Huang, 2008). However, there are not
94
many publications relating to genipin crosslinked nanoparticles (Choubey &
95
Bajpai, 2010; Maggi, Ciccarelli, Diociaiuti, Casciardi & Masci, 2011) which
96
encourages us the use of genipin as a crosslinking agent to develop chitosan
97
nanogels for biomedical applications.
98 99
Ac ce p
te
88
Since size distribution plays an important role in drug delivery application, it is necessary to prepare uniformly sized nanoparticles. However, the size
4 Page 4 of 34
distribution of chitosan nanoparticles is very difficult to control. Various studies
101
have shown that reverse microemulsion (w/o) is an effective way to prepare low
102
sized particles with uniform particle size distribution. This promising method for
103
the effective preparation of chitosan nanoparticles is a transparent, isotropic and
104
thermodynamically stable synthesis medium. Microemulsions not only act as
105
microreactors for the formation of nanoparticles but also inhibit excessive
106
aggregation of the forming particles (Liu, Shao, Gea & Chena, 2007). Several
107
authors have employed the well stablished microemulsion formulation based on
108
cyclohexane/Triton X100/n-hexane/water for obtaining chitosan nanosized
109
particles (Wang, Wang, Luo & Dai, 2008; Arteche Pujana, Pérez-Alvarez,
110
Cesteros Iturbe & Katime, 2012).
cr
us
an
M
111
ip t
100
In this work chitosan and genipin were chosen for preparing polymeric nanogels by crosslinking of chitosan. Both of these compounds display
113
advantageous biomedical properties. Nanogels were preparing by w/o
114
microemulsion method and their physicochemical properties, such as, particle
115
size, pH-sensitivity swelling, surface charge or water solubility, were studied.
117 118 119 120
te
Ac ce p
116
d
112
EXPERIMENTAL
Materials
Low molecular weight chitosan (Aldrich) was purified using the procedure
121
previously described (Signini & Campana, 1999). The deacetylation degree of
122
chitosan determined by 1H NMR was 79% which is in good agreement with the
123
value reported by the supplier (75-85%). Its viscosity average molecular weight
124
of chitosan measured by an Ubbelohde capillary viscometer (HAc 0.3M/NaAc
5 Page 5 of 34
0.2M, 25ºC) (Rinaudo, Milas & Dung, 1993) was 66,000 g/mol. Genipin, triton X-
126
100 and hexanol (for synthesis, 98%) were purchased from Sigma-Aldrich.
127
Cyclohexane (for synthesis, 98%), acetic acid (for analysis, 99.8%) and ethanol
128
(for analysis, 96%) were supplied from Panreac and D-glucosamine
129
hydrochloride by Acros Organics.
ip t
125
132
Preparation of nanogels
1g of chitosan was dissolved in 100 mL of 1%v/v aqueous acetic acid
us
131
cr
130
solution and separately genipin was dissolved in distilled water (1-540 mg/mL).
134
Chitosan nanoparticles were prepared using a procedure that was
an
133
previously described by Jia et al. (Jia, Yujun & Guangsheng, 2005) with minor
136
modification. Two separate W/O microemulsions were produced. The first
137
microemulsion (MC) was formed by dropwise addition of Triton X-100 to a
138
mixture of cyclohexane: n-hexanol: chitosan solution in ratio of 2.75:1:1
139
respectively. Triton X 100 was added until microemulsion became optically
140
transparent. The second microemulsion (MG) containing genipin solution but
141
without chitosan was prepared with the same procedure. Then, MG
142
microemulsion was added to MC microemulsion under magnetic stirring at
143
different crosslinker stoichiometric ratio. The crosslinking reaction was carried
144
out at 25 ºC for 4 days. During crosslinking reaction the microemulsion became
145
bluish. The microemulsion was broken by adding ethanol to obtain blue colored
146
nanoparticles. Nanogels were washed repeatedly with ethanol by centrifugation
147
(9000rpm, 10 min, at room temperature). Finally prepared nanogels were
148
washed with distilled water, ultrafiltered (OMEGA 76 MM 100K 12/PK) and
149
vacuum dried (55ºC, 24 hours).
Ac ce p
te
d
M
135
6 Page 6 of 34
150 151 152
Methods The nanogels were analyzed with NMR spectroscopy. 1H spectra were obtained on a Bruker Avance 500 MHz instrument and the samples were
154
dissolved in 2% w/w. CD3COOD/ D2O. Solid samples were analyzed by 13C
155
NMR spectroscopy with a Bruker Avance III 400 spectrometer.
cr
ip t
153
A Philips CM120 with Olympus SIS Morada digital camera transmission
157
electron microscope was used to characterize the size and morphology of the
158
dried chitosan nanoparticles. Nanoparticles were prepared dissolving dried
159
powder in 1%v/v aqueous acetic acid solution with a concentration of 1mg/mL.
160
which was then dried in a vacuum chamber.
163
an
M
Vis Spectrophotometer.
d
162
UV–Vis spectroscopy measurements were registered in a Cintra 303 UV-
The genipin-chitosan crosslinking reaction was characterized by UV-Vis.
te
161
us
156
Along the crosslinking reaction, spectra of the microemulsion samples were
165
obtained at different time intervals in a wavelength range from 200 to 700 nm.
166
Furthermore, the quantitative determination of the reacted free amino groups in
167
nanoparticles crosslinked with genipin was carried out by mean of the previous
168
calibration with D-glucosamine in the microemulsion medium. D-glucosamine
169
(50 to 750 mg/L) and genipin (500 mg/L) after being dissolved in the described
170
microemulsion medium were incubated at 25ºC. The absorbance at 605 nm was
171
measured and the obtained linear regression equation was A = 0.0188+
172
0.0021X (R2 = 0.99764).
Ac ce p
164
173
UV-Vis spectroscopy was also employed for the study of the water
174
solubility of the nanogels at different pH values by mean of the measurement of
7 Page 7 of 34
the transmittance of the nanogels dispersions at 750 nm. Nanogels were
176
dissolved in 1% HAc solution (1 mg/5 mL), and the transmittance was measured
177
24 hours after the pH of the solution was adjusted by the addition of 2 N NaOH
178
solution.
179
ip t
175
Dynamic laser light scattering measurements for determining the size of
the nanogels were performed using a Brookhaven BI-9000AT with a goniometer.
181
A water-cooled argon-ion laser was operated at 514.5 nm as the light source.
182
The time dependence of the intensity autocorrelation function of the scattered
183
intensity was obtained by using a 522 channel digital correlator. The size of the
184
nanoparticles was determined from the diffusion intensity of the particles using
185
the Stokes-Einstein equation. The size distribution was obtained by NNLS
186
analysis and the uncertainties of the measurements represented the standard
187
deviation of the mean of the replicate runs. The dried powder samples were
188
dispersed in 1% HAc solution (1mg/mL) during 3 days and then diluted until the
189
concentration was 40 mg/L. All measurements were made at room temperature.
190
The pH was changed with 2 N NaOH solution.
us
an
M
d
te
Ac ce p
191
cr
180
X-ray diffraction patterns were obtained by a Transmision STOE Stadip
192
X-ray diffractometer with a goniometer speed of 0.1º/90s. The range of
193
diffraction angle 2 was 2.5-34º.
194
Electrophoretic mobility measurements were performed with a Zeta-Sizer
195
IV (Malvern Instruments). Nanogels were dispersed in 1% HAc solution
196
(1mg/mL) during 3 days and finally diluted to a concentration of 40 mg/L. Data
197
were obtained from the average of at least ten measurements in universal cells.
198
The external pH was varied by adding 2 N NaOH solution.
199
8 Page 8 of 34
200
RESULTS AND DISCUSSION
201 202 1.- Study of the crosslinking reaction
ip t
203 204
Covalently crosslinked chitosan nanoparticles were prepared within a
cr
205
reverse microemulsion medium in order to control the colloidal particle size of
207
the prepared nanogels. This modification reaction took place when separately
208
prepared chitosan and genipin microemulsion were mixed as described above.
209
This crosslinking occurs by mean of a complex mechanism which is believed
210
that consists in two reactions involving different sites on the genipin molecule
211
(Butler, Ng & Pudney, 2003) and produces dark-blue coloration in the reaction
212
mixture. The appearance of this coloration in the reaction mixture of genipin and
213
chitosan is traditionally considered as rapid evidence that the crosslinking
214
reaction is taking place (Butler, Ng & Pudney, 2003; Muzzarelli, 2009).
an
M
d
te
The evolution of the crosslinking reaction was monitored by mean of the
Ac ce p
215
us
206
216
UV-Vis spectra of the final microemulsion. As can be seen in Figure 1A, a new
217
peak appeared at 605 nm, which corresponds to the formed blue pigment, and
218
continued to increase in intensity throughout the course of the reaction.
219
Ramos et al. (Ramos-Ponce, Vega, Sandoval-Fabián, Colunga-Urbina,
220
Balagurusamy, Rodriguez-Gonzalez & Contreras-Esquivel, 2010) described an
221
efficient colorimetric method using genipin as a specific reagent for quantitative
222
determination of free amino groups of chitin derivatives. Following this
223
procedure, the relationship between the absorbance of the microemulsion at 605
224
nm and the concentration of free amino groups in the nanogels was determined
9 Page 9 of 34
by mean of a calibration curve with standard solutions of D-glucosamine. The
226
method reported by Ramos et al. could be applied to the microemulsion medium
227
after slight modifications. The concentration of modified free amino groups as a
228
function of reaction time was analyzed in order to optimize the reaction time.
229
Results of this study are shown in Figure 1B, demonstrating that after 7 days the
230
end point of the reaction was reached.
cr
ip t
225
231 3.- NMR characterization
us
232 233
The success of the modification reaction was also confirmed and
an
234
quantitatively evaluated by 1H NMR. Pure chitosan shows a peak at 2.0–2.1
236
ppm corresponding to the three protons of N-acetyl glucosamine (GlcNAc) and a
237
peak at 3.1–3.2 ppm due to H-2 proton of glucosamine (GlcN) residues which
238
represent the primary amino group content of the chitosan. Figure 2 displays the
239
spectra of several genipin-chitosan nanogels. A clear decrease of the signal at
240
3.2 ppm was observed for all the samples, indicating the loss of amine groups
241
by reaction with genipin showing that the addition of genipin to chitosan was
242
successfully carried out.
d
te
Ac ce p
243
M
235
The peak of the 1H NMR spectrum of chitosan corresponding to the
244
methyl protons at 2.0 - 2.1 ppm possess the highest resolution (Kasaai, 2010)
245
and was used for the determination of the free amino content by mean of the
246
integration of the resonance of H-2 protons at 3.2 ppm. Thus, the modification
247
degree of the chitosan chains was evaluated by the decrease of the
248
glucosamine signal (3.2 ppm). The crosslinking grade was calculated assuming
249
that the reaction occurs on both reaction sites of the genipin molecule. Table 1
10 Page 10 of 34
lists the nanogels compositions obtained by 1H NMR analysis for each initial
251
reaction feed. The data collected in Table 1 show a rather good correlation
252
between genipin stoichiometric ratio (with respect to the total amount of
253
glucosamine units) in initial reaction mixture and final modification degree in the
254
nanogels, especially for intermediate compositions. These results suggest that
255
the linkage genipin-chitosan occurs in a ratio around 1:2 and therefore the self-
256
polymerization of genipin is not likely to take place. However, for high crosslinker
257
content, the ratio genipin feed: modified chitosan increased. Assuming that the
258
reaction yields did not varied significantly, it could be related to a higher
259
probability that polymerization takes place. This in accordance with an
260
intermediate brownish colour appeared during crosslinking reaction of these
261
samples before the blue intense coloration (Mi, Shyu & Peng, 2005).
cr
us
an
M
262
ip t
250
On another hand, modification degrees of chitosan in the nanogels obtained by 1H NMR analysis agreed with those obtained by UV-Vis
264
spectroscopy (Table 1).
te
Besides, 13C NMR spectroscopy also corroborates the modification of
Ac ce p
265
d
263
266
linear chitosan with genipin. Figure 3 shows the 13C NMR of initial pure chitosan
267
and chitosan crosslinked with genipin. The crosslinking of chitosan with genipin
268
results in some slight changes on the 13 C NMR spectrum of chitosan. The
269
resonance signal of chitosan C-4 at 81-87 ppm decreased and becomes into a
270
shoulder. Mi et al. (Mi, Sung & Shyu, (2000); Mi, Sung & Shyu, 2001) attributed
271
this fact to a conformational change from the linear structure of original chitosan
272
to crosslinked chitosan, revealing the formation of a secondary amide and
273
heterocyclic amino linkage, and so, the crosslinking of chitosan. Additionally, a
274
slight shift of C-1 resonance from 112 ppm to a broad resonance centered at
11 Page 11 of 34
275
around 109 ppm occurs, which is attributed to the incorporation of the
276
heterocyclic ring at C-2 (Mi, Sung & Shyu, 2000).
277
279
4.- Water solubility
ip t
278
The applications of chitosan nanoparticles suffer severe limitations
because they are no dispersible in neutral or alkaline pH aqueous media. This
281
water insolubility in high pH solutions is due to the presence of rigid crystalline
282
domains, formed by intra- and/or intermolecular hydrogen bonding (Nishimura,
283
Kohgo, Kurita & Kuzuhar, 1991).
us
The solubility of the prepared genipin-chitosan nanogels was
an
284
cr
280
characterized as a function of pH and compared with the unmodified chitosan.
286
Figure 4A shows the transmittance at 750 nm of aqueous solutions of linear
287
chitosan and genipin-chitosan nanogels prepared in the above described
288
conditions.
d te
289
M
285
In general, as the pH decreases the protonation of free amino groups of chitosan chains takes place and the H-bonding interactions decreases,
291
improving water-polymer interaction and consequently increasing the
292
transmittance of aqueous dispersions (Figure 4A). So, when pH increases the
293
dispersions become opaque and particles aggregation takes places.
294
Ac ce p
290
The solubility of the nanogels was determined from pH50 parameter that is
295
defined as the pH value when the transmittance at 750 nm reached 50%.
296
Loosely crosslinked nanogels (< 7.5 mol. % genipin) showed a slightly improved
297
stability with the pH, displaying a value of 7.3 for the pH50 parameter, while the
298
value measured for pure chitosan was 7.0. When the genipin content was higher
12 Page 12 of 34
299
than 7.5 mol. % the pH50 of the nanogels was lower than that of the pure
300
chitosan in all the cases.
301
It is well kwon that the modification of chitosan with more or less bulky pendant groups leads to the reduction of hydrogen bonding in
303
chitosan and therefore, the solubility of the chitosan derivatives increases. In this
304
regard, the increase in water solubility of PEGylated chitosan (Chan, Kurisawa,
305
Chung & Yang, 2007) has been attributed to the decrease of intermolecular
306
interactions, as well as the swelling of N-alkyl and N-acylated chitosans in
307
water, in spite of their hydrophobicity (Ravi Kumar, 2000; Shasiwa, Kawasaki,
308
Nakayama, Muraki, Yamamoto & Aiba, 2002).
cr
us
an
In general, chitosan derivatives that precipitate in basic medium have a
M
309
ip t
302
high degree of crystallinity. On the contrary, chitosan obtained with amorphous
311
form and loosely aggregated state is soluble in water (Lu, Song, Cao, Chen &
312
Yao, 2004).
te
d
310
Figure 4B shows the X-ray diffractograms of some of the samples whose
314
solubility behaviour is displayed in Figure 4A. Pure chitosan sample displays the
315
typical pattern of chitosan substrates with peaks at 2 around 11 and 20º. As
316
can be observed, the chemical crosslinking with genipin causes the loss of
317
crystallinity in the samples. All the studied genipin-chitosan nanogels displayed
318
a difractogram of an amorphous material.
319
Ac ce p
313
Thus, it could be concluded that a moderate crosslinking disturbs the intra
320
and inter intermolecular hydrogen bonding resulting in the observed
321
enhancement in solubility. However, as crosslinking increases (up to 7.5 mol.
322
%), in spite of the weakened intra-intermolecular interactions of chitosan chains,
13 Page 13 of 34
323
the nanogels become more and more compact and a close and rigid network is
324
generated, leading to a substantial reduction of the water solubility.
325 5.- Particle size and swelling properties of the nanogels
ip t
326 327
The morphology and particle size distribution of dried nanogels was
cr
328
studied by TEM. TEM images (Figure 5A) indicate that genipin-chitosan
330
nanogels are spherical, have nearly uniform particle size distribution (Figure 5B)
331
and there is no severe particle agglomeration, which are very interesting
332
qualities for biomedical applications, such as drug delivery. The average
333
diameters of nanoparticles ranged from 3 to 20 nm.
M
an
us
329
The particle size of swollen nanogels was determined by QELS
335
measurements. Due to the adhesive nature of chitosan in aqueous solution,
336
these nanoparticles tend to form aggregates that lead to a higher average
337
hydrodynamic diameter than the actual size of the particles (Wu, Zhou & Wang,
338
1995). The progressive dilution of the nanoparticle dispersions has been
339
reported as an effective way to reduce the inter-particle interaction and the
340
presence of large aggregates of chitosan is (Sorlier, Rochas, Morfin, Viton &
341
Domard, 2003) and was applied in this study. However, the solubility study
342
revealed in comparison with pure chitosan a higher tendency of genipin-chitosan
343
nanogels to aggregate, except for loosely crosslinked ones (7.5 mol. %).
344
Ac ce p
te
d
334
Figure 6 shows the average diameters of the nanogels determined by
345
QELS at pH = 4.0 for several genipin stoichiometric ratios. No strong correlation
346
was found between the hydrodynamic diameter of the genipin-chitosan nanogels
347
and the crosslinking degree. Besides, as is shown in Figure 7, a slight pH-
14 Page 14 of 34
348
responsive behaviour was observed. Thus, it could be concluded that the
349
nanogels display a weak swelling capacity due to the rigid and hydrophobic
350
nature of the employed crosslinker. The swelling effect induced in an ionic network, such as chitosan
352
nanogels, can be considered as the accumulative effect of the mixing, elastic
353
and ionic contributions (Flory, & Rehner, 1943). In the case of the genipin-
354
chitosan nanogels at pH = 4, an increase in crosslinking has opposite effects.
355
On the one hand leads to the diminishing of ionic contribution due to the
356
decrease of ionizable groups in the network, and to the enhancement of the
357
elastic force. Both of these effects tend to limit the swelling of the gel. However,
358
as crosslinker concentration increases (up to 7.5 mol. %), the mixing
359
contribution is drastically reduced, due to the hydrophobic and closed nature of
360
the network, and aggregation takes place, increasing the measured
361
hydrodynamic diameter. This may explain that the determined particle sizes of
362
the nanoparticles varying the content of genipin were fairly similar each other
363
(Figure 6).
cr
us
an
M
d
te
Ac ce p
364
ip t
351
At constant degree of crosslinking, when the external pH was varied it
365
was found that the nanoparticles swell slightly at pH below 5.5 (Figure 7). This
366
may be due to the protonation of free amine groups of chitosan chains at low
367
pH, which results in an electrostatic repulsion charge favouring the penetration
368
of water into the gel. For basic pHs the precipitation of the nanogels takes place
369
and particle size or could not be measured or increased as consequences of
370
particle aggregation. Studies of the pH-responsive swelling of genipin-chitosan
371
macrogels also did not displayed an abrupt phase transition (Kaminski, K.,
372
Zazakowny, K., Szczubiałka, K., Nowakowska M., 2008) that was observed in
15 Page 15 of 34
other studied chitosan networks (Arteche Pujana, Pérez-Alvarez, Cesteros
374
Iturbe & Katime, 2012). In this sense, Jharen et al. (Jahren, Butler, Adams &
375
Cameron, 2010) found that the pH-responsive phase transition decreased with
376
crosslinking in genipin-chitosan hydrogels and disappeared completely for a
377
genipin percentage of 80 mol. %.
To obtain information about the electrical state of the ionizable groups
cr
378
ip t
373
responsible of the pH-responsive behaviour of the nanoparticles, zeta potential
380
of genipin-chitosan nanogels was measured as function of genipin content
381
(Figure 6), as well as, of pH (Figure 7). In all the samples the value of the zeta
382
potential increased up to 20 mV when the pH increased from 4 to 9. All the
383
nanogels showed positive charge at acidic conditions, so all of them maintained
384
the chitosan mucoadhesive and absorption enhancement potential properties.
385
It is shown in Figure 6 that the zeta potential values of protonated
386
networks (pH = 4.0) decreased as the nanogels crosslinking was higher, proving
387
the loss of free amine moiety along the chitosan backbone within the
388
modification with genipin. Regarding pH (Figure 7), as the chitosan chains
389
contain free amine pH-ionizable groups, a pH variation modifies the electrical
390
state of the network. The amine primaries groups are protonated in acidic media,
391
and the surface charge is positive and increases along the ionization process
392
occurs. So, the progressive protonation of amino groups as pH decreases was
393
confirmed by measuring the electrokinetic potential of the nanogels.
Ac ce p
te
d
M
an
us
379
394 395
CONCLUSIONS
396
16 Page 16 of 34
397
Chitosan low-size nanogels were prepared by the combination of two well-known methods such as, reverse microemulsion and crosslinking by
399
genipin. Nanogels sizes ranging from 3-20 nm determined by TEM, and 270-390
400
nm determined by QELS in aqueous media, could be obtained by this covalent
401
crosslinking method. Loosely crosslinked nanogels displayed an improved water
402
solubility compared with pure chitosan. The variation of water solubility of
403
chitosan due to the crosslinking with genipin could be consider as a compromise
404
between the decrease of crystallinity and the elastic force within the generated
405
network. This elastic contribution may play an essential role in the water
406
solubility and pH-sensitive swelling of the nanogels due to the low molecular
407
weight and rigid nature of genipin molecule.
M
an
us
cr
ip t
398
408 409
ACKNOWLEDGEMENTS
413 414 415 416 417
te
412
Financial support for this work was provided by the Basque Government and the University of the Basque Country (UPV/EHU), for awarding a fellowship.
Ac ce p
411
d
410
REFERENCES
Agnihotri, S. A., Mallikarjuna, N. N. & Aminabhavi, T. M. (2004). Recent
418
advances on chitosan-based micro- and nanoparticles in drug delivery.
419
Journal of Controlled Release, 100, 5–28.
420 421
Alonso, M. J. & Sánchez, A. (2003). The potential of chitosan in ocular drug delivery. Journal of Pharmacy and Pharmacology, 55, 1451–1463.
17 Page 17 of 34
422
Arteche Pujana, M., Pérez-Alvarez, L., Cesteros Iturbe, L. C. & Katime, I.
423
(2012). Water dispersible pH-responsive chitosan nanogels modified with
424
biocompatible crosslinking-agents. Polymer, 53, 3107-3116. Banerjee, T., Mitra, S., Kumar Singh, A., Kumar Sharma, R. & Maitra, A. (2002).
ip t
425
Preparation, characterization and biodistribution of ultrafine chitosan
427
nanoparticles. International Journal of Pharmaceutics, 243, 93–105.
428
cr
426
Butler, M.F., Ng, Y.F. & Pudney, P.D.A. (2003). Mechanism and kinetics of the crosslinking reaction between biopolymers containing primary amine groups
430
and genipin. Journal of Polymer Science. Part A: Polymer Chemistry, 41,
431
3941–3953
an
Chan, P., Kurisawa, M., Chung, J. E & Yang, Y. Y. (2007). Synthesis and
M
432
us
429
characterization of chitosan-g-poly(ethylene glycol)-folate as a non-viral
434
carrier for tumor-targeted gene delivery. Biomaterials, 28, 540-549.
d
433
Choubey, J. & Bajpai, A. K. (2010). Investigation on magnetically controlled
436
delivery of doxorubicin from superparamagnetic nanocarriers of gelatin
437
crosslinked with genipin. Journal of Materials Science- Materials in Medicine,
439 440 441 442 443 444 445
Ac ce p
438
te
435
21, 1573–1586.
Crini, G. (2005). Recent developments in polysaccharide-based materials used as adsorbents in wastewater treatment. Progress in Polymer Science 30, 38– 70.
Felt, O., Buri, P. & Gurny, R. (1998). Chitosan: a unique polysaccharide for drug delivery. Drug Development and Industrial Pharmacy, 24, 979– 993. Flory, P. J. & Rehner, J. (1943). Statistical Mechanics of Cross-Linked Polymer Networks II. Swelling. Journal of Chemical Physics, 11, 521.
18 Page 18 of 34
446
González, A., Strumia, M. C. & Alvarez Igarzabal, C. I. (2011). Cross-linked soy
447
protein as material for biodegradable films: synthesis, characterization and
448
biodegradation. Journal of Food Engineering, 106, 331–338
450
Illum, L., Farraj, N.F. & Davis, S.S. (1994). Chitosan as a novel nasal delivery
ip t
449
system for peptide drugs. Pharmaceutical Research, 11, 1186– 1189.
Jahren, S. L., Butler, M. F., Adams, S. & Cameron, R. E. (2010). Swelling and
452
Viscoelastic Characterisation of pH-Responsive Chitosan Hydrogels for
453
Targeted Drug Delivery. Macromolecular Chemistry and Physics, 211, 644–
454
650.
us
Jia, Z., Yujun, W. & Guangsheng L. (2005). Adsorption of diuretic furosemide
an
455
cr
451
onto chitosan nanoparticles prepared with a water-in-oil nanoemulsion
457
system. Reactive & Functional Polymers, 65, 249–257. Jin, J., Song, M. & Hourston, D. J. (2004). Novel chitosanbased films cross-
d
458
M
456
linked by genipin with improved physical properties. Biomacromolecules, 5,
460
162-168.
te
459
Kaminski, K., Zazakowny, K., Szczubiałka, K. & Nowakowska M. (2008). pH-
462
sensitive genipin-cross-linked chitosan microspheres for heparin removal.
463
Ac ce p
461
Biomacromolecules, 9, 3127–3132.
464
Kasaai, M. R. (2010). Determination of the degree of N-acetylation for chitin and
465
chitosan by various NMR spectroscopy techniques: A review. Carbohydrate
466
Polymers, 79, 801–810.
467
Lee, J.-Y., Nam, S.-H., Im, S.-Y., Park, Y.-J., Leeb, Y.-M., Seol Y.-J., Chung, C.-
468
P. & Lee S.-J. (2002). Enhanced bone formation by controlled growth factor
469
delivery from chitosan-based biomaterials. Journal of Controlled Release, 78,
470
187–197.
19 Page 19 of 34
471
Liu, B. S. & Huang, T. B. (2008). Nanocomposites of Genipin-Crosslinked
472
Chitosan/Silver Nanoparticles–Structural Reinforcement and Antimicrobial
473
Properties. Macromolecular Bioscience, 8, 932–941.
476
ip t
475
Liu, G., Shao, L., Gea, F. & Chena, J. (2007). Preparation of ultrafine chitosan particles by reverse microemulsion. China Particuology, 5, 384–390.
Liu, W.G. & Yao, K.D. (2002). Chitosan and its derivatives--a promising non-viral
cr
474
vector for gene transfection. Journal of Controlled Release, 83, 1– 11.
478
Lu, S., Song, X., Cao, D., Chen, Y. & Yao K. (2004). Preparation of Water-
479
us
477
Soluble Chitosan. Journal of Applied Polymer Science, 91, 3497–3503. Maggi, F., Ciccarelli, S., Diociaiuti, M., Casciardi, S. & Masci G. (2011). Chitosan
481
Nanogels by Template Chemical Cross-Linking in Polyion Complex Micelle
482
Nanoreactors. Biomacromolecules, 12, 3499−3507
M
an
480
Malhotra, M., Kulamarva, A., Sebak, S., Paul, A., Bhathena, J., Mirzaei M. &
484
Prakash, S. (2009). Ultrafine chitosan nanoparticles as an efficient nucleic
485
acid delivery system targeting neuronal cells. Drug Development and
486
Industrial Pharmacy 35, 719–726.
Ac ce p
te
d
483
487
Mi, F.-L., Sung, H.-W. & Shyu, S.-S. (2000). Synthesis and characterization of a
488
novel chitosan-based network prepared using naturally occurring crosslinker.
489 490 491
Journal of Polymer Science- Part A: Polymer Chemistry, 38, 2804–2814.
Mi, F.-L. Sung, H.-W. & Shyu; S.-S. (2001). Release of Indomethacin from a Novel Chitosan Microsphere Prepared by a Naturally Occurring Crosslinker:
492
Examination of Crosslinking and Polycation–Anionic Drug Interaction. Journal
493
of Applied Polymer Science, 81, 1700–1711.
494 495
Mi, F.-L., Shyu, S.-S. & Peng, C.-K. (2005). Characterization of ring-opening polymerization of genipin and pH-dependent cross-linking reactions between
20 Page 20 of 34
496
chitosan and genipin. Journal of Polymer Science- Part A: Polymer
497
Chemistry, 43, 1985–2000.
498
Miyazaki, S. Nakayama A, Oda M, Takada M & Attwood D. (1994). Chitosan and sodium alginate based bioadhesive tablets for intraoral drug delivery.
500
Biological & Pharmaceutical Bulletin, 17, 745-7.
503 504
cr
502
Muzzarelli, R.A.A., Jeuniaux, C. & Gooday, G. W. (1986). Chitin in nature and Technology. New York: Plenum Press.
Muzzarelli, RAA. (2009). Genipin-crosslinked chitosan hydrogels as biomedical
us
501
ip t
499
and pharmaceutical aids. Carbohydrate Polymers, 77, 1-9.
Muzzarelli, R.A.A., Boudrant, J., Meyer, D., Manno, N., DeMarchis, M. &
506
Paoletti, M.G. (2012). Current views on fungal chitin/chitosan, human
507
chitinases, food preservation, glucans, pectins and inulin: A tribute to Henri
508
Braconnot, precursor of the carbohydrate polymers science, on the chitin
509
bicentennial. Carbohydrate Polymers, 87, 995-1012.
512 513 514 515 516 517
M
d
te
511
Muzzarelli, R.A.A., Greco, F., Busilachi, A., Sollazzo, V. & Gigante, A. (2012). Chitosan, hyaluronan and chondroitin sulfate in tissue engineering for
Ac ce p
510
an
505
cartilage regeneration: a review. Carbohydrate polymers, 89, 723-739.
Nishimura, S.I., Kohgo, O., Kurita, K. & Kuzuhara, H. (1991). Chemospecific manipulations of a rigid polysaccharide: syntheses of novel chitosan derivatives with excellent solubility in common organic solvents by regioselective chemical modifications. Macromolecules, 24, 4745–4748.
Ramos-Ponce, L. M, Vega, M., Sandoval-Fabián, G. C., Colunga-Urbina, E.,
518
Balagurusamy, N., Rodriguez-Gonzalez, F. J. & Contreras-Esquivel, J, C.
519
(2010). A Simple Colorimetric Determination of the Free Amino Groups in
21 Page 21 of 34
520
Water Soluble Chitin Derivatives Using Genipin. Food Science &
521
Biotechnology, 19, 683-689.
524 525 526
Reactive & Functional Polymers, 46, 1–27.
ip t
523
Ravi Kumar, M.N.V. (2000). A review of chitin and chitosan applications.
Ravi Kumar, M.N.V. (2004). Chitosan chemistry and pharmaceutical perspectives. Chemical Reviews, 104, 6017–6084.
cr
522
Rinaudo, M., Milas, M. & Dung, P.L. Characterization of chitosan. Influence of ionic strength and degree of acetylation on chain expansion. (1993).
528
International Journal of Biological Macromolecules, 15, 281-285.
531 532
an
European Polymer Journal, 46, 1537-1544.
M
530
Rinaudo, M. (2010). New way to crosslink chitosan in aqueous solution.
Shahidi, F., Arachchi, J. K. V. & Jeon, Y.-J. (1999). Food applications of chitin and chitosans. Trends in Food Science & Technology, 10, 37-51.
d
529
us
527
Shasiwa, H., Kawasaki, N., Nakayama, A., Muraki, E., Yamamoto, N. & Aiba, S.
534
(2002). Chemical modification of Chitosan. 14: Synthesis of water-soluble
535
chitosan derivatives by simple acetylation. Biomacromolecules, 3, 1126-1128.
537 538 539 540 541
Ac ce p
536
te
533
Signini, R. & Campana Filho S. P. (1999). On the preparation and characterization of chitosan hydrochloride. Polymer Bulletin, 42, 159–166.
Sung , H. W., Huang, R. N., Huang, L. L. & Tsai, L. L. (1999). In vitro evaluation of cytotoxicity of a naturally occurring cross-linking reagent for biological tissue fixation. Journal of Biomaterials Science, Polymer Edition, 10, 63-78.
Sorlier, P., Rochas, C, Morfin, I., Viton, C. & Domard, A. (2003). Light Scattering
542
Studies of the Solution Properties of Chitosans of Varying Degrees of
543
acetylation. Biomacromolecules, 4, 1034-1040.
22 Page 22 of 34
544
Wang, Y., Wang, X., Luo, G. & Dai, Y. (2008). Adsorption of bovin serum
545
albumin (BSA) onto the magnetic chitosan nanoparticles prepared by a
546
microemulsion system. Bioresource Technology, 99, 3881–3884. Wenling, C., Mingyu, C., Qiang, A., Yandao, G., Nanming, Z. & Xiufang, Z.
ip t
547
(2005). Physical, mechanical and degradation properties, and Schwann cell
549
affinity of cross-linked chitosan films. Journal of Biomaterials Science,
550
Polymer Edition, 16, 791-807.
us
552
Wu, C., Zhou, S. & Wang, W. (1995). A dynamic laser light-scattering study of chitosan in aqueous solution. Biopolymers, 35, 385-392.
an
551
cr
548
553
M
554 555
559 560 561 562 563 564
te
558
Ac ce p
557
d
556
565 566
23 Page 23 of 34
FIGURE CAPTIONS
FIGURE 1. Evolution of A) the visible absorption spectrum of chitosan
ip t
microemulsion during crosslinking reaction with genipin and B) modification degree of chitosan with crosslinking reaction time for: ○) 7.5, □) 12.5, ●) 18.8, ■)
cr
37.5 mol. % of genipin.
us
FIGURE 2. 1H NMR spectrum of genipin-chitosan nanogels (7.5 mol. % of genipin) and amplified resonance signal corresponding to the Ha protons
an
(chitosan H-2 of GlcN ) of nanogels crosslinked with different genipin feeds (0,
M
7.5, 15 and 50 mol. %).
d
FIGURE 3. 13 C NMR spectrum of (A) chitosan nanoparticles crosslinked with
te
genipin and (B) pure chitosan.
Ac ce p
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.
24 Page 24 of 34
FIGURE 6. (□) Zeta potential (●) and Particle size determined by QELS for genipin-chitosan nanogels at pH = 4.0 as function of crosslinker content.
ip t
FIGURE 7. (●) Average hydrodynamic diameter and (○) zeta potential of
Ac ce p
te
d
M
an
us
cr
nanogels as function of external pH (for 12.5 mol. % crosslinker).
25 Page 25 of 34
HIGHLIGHTS
Chitosan was crosslinked with genipin in reverse microemulsion medium.
ip t
Chitosan-genipin crosslinking reaction was quantitatively evaluated by UV-Vis and 1H NMR.
cr
Chitosan-genipin nanogels showed improved water solubility at neutral pHs.
Swelling behaviour of the nanogels was studied as function of crosslinking
Ac ce p
te
d
M
an
us
degree and pH.
26 Page 26 of 34
TABLES
TABLE 1. Modification degree of obtained genipin-chitosan nanogels analyzed by 1H
Genipin Feed
UV-Vis
cr
H NMR
Modification Crosslinking Modification Crosslinking
us
1
ip t
NMR and UV-Vis spectroscopy.
(stoichiometric
degree
mol.%)
(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
(mol. %)
degree
(mol. %)
Ac ce p
te
d
M
an
(mol. %)
27 Page 27 of 34
Ac
ce
pt
ed
M
an
us
cr
i
Figure1
Page 28 of 34
Ac
ce
pt
ed
M
an
us
cr
i
Figure 2
Page 29 of 34
Ac
ce
pt
ed
M
an
us
cr
i
Figure 3
Page 30 of 34
Ac ce p
te
d
M
an
us
cr
ip t
Figure 4
Page 31 of 34
Ac
ce
pt
ed
M
an
us
cr
i
Figure 5
Page 32 of 34
Ac
ce
pt
ed
M
an
us
cr
i
Figure 6
Page 33 of 34
Ac
ce
pt
ed
M
an
us
cr
i
Figure 7
Page 34 of 34