ARTICLE IN PRESS

G Model
IJP 13750 19

International Journal of Pharmaceutics xxx (2013) xxxxxx

Contents lists available at ScienceDirect

International Journal of Pharmaceutics

journal homepage: www.elsevier.com/locate/ijpharm

Synthesis and characterization of a novel controlled release zinc

oxide/gentamicinchitosan composite with potential applications in
wounds care

4
5
6
7

Q1

Bogdan Stefan Vasile a , Ovidiu Oprea a, , Georgeta Voicu a , Anton Ficai a ,

Ecaterina Andronescu a , Andrei Teodorescu a , Alina Holban b
a
b

University Politehnica of Bucharest, Faculty of Applied Chemistry and Materials Science, Gh. Polizu 1, Bucharest, Romania
University of Bucharest, Faculty of Biology, Department of Microbiology and Immunology, Intr. Portocalelor 1-3, Bucharest, Romania

a r t i c l e

i n f o

a b s t r a c t

10
11
12
13
14

Article history:
Received 15 August 2013
Received in revised form 3 November 2013
Accepted 18 November 2013
Available online xxx

15

22

Keywords:
Zinc oxide
Chitosan
Gentamicin
Controlled release
Improved wound dressing
Antimicrobial effect

23

1. Introduction

16
17
18
19
20
21

24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39

Freshly prepared ZnO nanoparticles were incorporated into a chitosan solution in weight ratios ranging
from 1:1 to 12:1. Starting from the ratio of 3:1 the chitosan solution was transformed into a gel with a high
consistency, which incorporates 15 mL water for only 0.1 g solid substance. The powders obtained after
drying the gel were characterized by X-ray diffraction (XRD), transmission electron microscopy (TEM) and
thermal analysis (TG-DSC). The electronic (UVvis), infrared (FTIR) and photoluminescence (PL) spectra
were also recorded. ZnO particles were coated with gentamicin and incorporated into the chitosan matrix,
to yield a ZnO/gentamicinchitosan gel. The release rate of gentamicin was monitored photometrically.
This ZnO/gentamicinchitosan gel proved great antimicrobial properties, inhibiting Staphylococcus aureus
and Pseudomonas aeruginosa growth in both planktonic and surface-attached conditions. The results
indicate that the obtained composite can be used in cutaneous healing for developing improved wound
dressings, which combine the antibacterial activity of all three components with the controlled release
of the antibiotic. This wound dressing maintains a moist environment at the wound interface, providing
a cooling sensation and soothing effect, while slowly releasing the antibiotic. The system is fully scalable
to any other soluble drug, as the entire solution remains trapped in the ZnOchitosan gel.
2013 Published by Elsevier B.V.

Beside its various technical applications (Vaja et al., 2012, 2013),

zinc oxide may be also used in a series of cosmetic products, sun
protection cream, acne treatment or for wound dressings. Application of zinc oxide has been shown to accelerate the healing of
both chronic and acute wounds and it also exhibits antibacterial,
antifungal and anti-inammatory properties (Selvam et al., 2012;
Subhasree et al., 2012; Vlad et al., 2012). ZnO effect on wounds
epithelialization as well as its bacteriostatic properties promotes
it as an effective ingredient for topical wound dressings. It can be
used in the treatment of various dermatitis, diaper rashes, diaper
wipes, blisters, and open skin sores (Kumar et al., 2012a; Shalumon
et al., 2011).
Chitosan, an abundant natural polysaccharide well known for its
biodegradable and non-toxic properties, is popular in the eld of
biomedicine due to its interesting physicochemical properties and
potential for a wide range of applications (Casettari et al., 2012;

Corresponding author. Tel.: +40 724697047.

E-mail address: ovidiu73@yahoo.com (O. Oprea).

Grumezescu et al., 2012a,b; Sogias et al., 2012). One of the applications that received much attention in the last years is related
to the potential uses of chitosan in the process of wound healing (Archana et al., 2013; Ribeiro et al., 2013; Wijekoon et al.,
2013).
Incorporation of zinc oxide nanoparticles into chitosan comes
as one of natural solutions to develop new bandages for wound
dressing (Kumar et al., 2012a,b), as the antibacterial activity of the
composite ZnOchitosan is also well documented in the literature
(El Shafei and Abou-Okeil, 2011; Liu and Kim, 2012; Perelshtein
et al., 2013).
Gentamicin belongs to the class of aminoglycoside antibiotics,
widely used to treat many types of serious infections, particularly
those caused by Gram-negative bacteria. It is usually used to treat
skin infections, soft tissues and bone infection, and also extreme
burns-associated infections (Builders et al., 2013; Manjunatha et al.,
2010). Rapid debridement of burn wounds and the application of
effective topical and systemic antimicrobial agents can improve the
efcacy of burn therapy (Sun et al., 2012).
Many types of nosocomial pathogens are drug-resistant and
patients infected with these drug-resistant pathogens are at a high
risk. As different bacterial strains become increasingly resistant

0378-5173/$ see front matter 2013 Published by Elsevier B.V.

http://dx.doi.org/10.1016/j.ijpharm.2013.11.035

Please cite this article in press as: Vasile, B.S., et al., Synthesis and characterization of a novel controlled release zinc oxide/gentamicinchitosan
composite with potential applications in wounds care. Int J Pharmaceut (2013), http://dx.doi.org/10.1016/j.ijpharm.2013.11.035

40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61

G Model
IJP 13750 19

ARTICLE IN PRESS
B.S. Vasile et al. / International Journal of Pharmaceutics xxx (2013) xxxxxx

Fig. 1. The ZnOchitosan gels. On top row (from left to right) synthesis with ZnO:chitosan ratio of 1:1; 2:1; 3:1; 4:1; 5:1 and 6:1. On bottom row, the ZnOchitosan gels have
high consistency starting with ZnO:chitosan ratio 3:1.

nanopowders and chitosan solution in acetic acid 1%, as follows.

0.0250 g chitosan were dissolved in 10 mL acetic acid solution (1%).
Freshly prepared ZnO nanoparticles (from 0.0250 g up to 0.3000 g)
were suspended in 5 mL bi-distilled water and ultrasonicated for
5 min. The milky ZnO suspension was quickly added under magnetic stirring over the chitosan solution. For the formulation with
weight ratio ZnO/chitosan of 1:1 and 1:2 a viscous, clear solution is
obtained. For the formulation with weight ratio ZnO/chitosan 3:1,
the ZnOchitosan gel (opalescent-white) is formed in few seconds,
and the magnetic bar is immobilized inside the gel (Fig. 1). The
ZnOchitosan gel can be extracted from the baker, and cut in any
shape, just like rubber. It is stable up to three days in laboratory
atmosphere, but can be kept over three months in water, with no
further swelling (Fig. 2).
For
the
synthesis
of
ZnO/gentamicinchitosan
gel,
ZnO/gentamicin hybrid was obtained starting from 0.3000 g
ZnO and 5 mL gentamicin solution (0.0500 g gentamicin). The 5 mL
aqueous solution containing the ZnO/gentamicin nanopowder was
added over 10 mL CH3 COOH solution 1% of 0.0250 g chitosan.
The ZnOchitosan and ZnO/gentamicinchitosan nanopowders
were obtained by drying the appropriate gel and ne grinding the
lms that result.

85

to antibiotics, the search for new strategies to deal with resistant

bacteria is emerging (Grumezescu et al., 2012c). Researchers
need to identify and develop next generation drugs or agents
to control bacterial infections (Grumezescu et al., 2012d). The
main strategy is to combine different antibacterial agents to
overcome the resistance of the microorganisms and to obtain
synergic antibacterial activity (Andronescu et al., 2012; Saviuc
et al., 2011; Song et al., 2013; Zabielinski et al., 2013). Controlled
release and targeting of drugs is another modern approach that is
trying to improve the efciency of various substances (Eidi et al.,
2012; Grumezescu et al., 2012e; Mihaiescu et al., 2011, 2012). In
a previous study (Voicu et al., 2013) we proved that gentamicin
coated ZnO nanoparticles can be successfully used to design a large
spectrum-active antibacterial wound dressing material composed
by synergistically acting components.
The aim of this study was to develop a newly ZnOchitosan
based controlled release nanosystem, with great absorptive properties, for improving the efciency of antibiotics in external
infections. This material may be successfully used in wound care,
the potential applications being derived from its enhanced antibacterial activity, along with the high water content that helps in
maintaining a moist environment at the wound interface, providing a cooling sensation and soothing effect, while slowly releasing
the antibiotic, on the other side.

86

2. Materials and methods

87

2.1. Experimental procedure

(a) Electron microscope images. The transmission electron images

were obtained on dried, nely powdered samples using a
TecnaiTM G2 F30 S-TWIN high resolution transmission electron microscope from FEI, operated at an acceleration voltage
of 300 kV obtained from a Schottky eld emitter with a TEM

point resolution of 2 A and line resolution of 1.02 A.

(b) X-ray diffraction. X-ray powder diffraction patterns were
obtained with a Shimadzu XRD6000 diffractometer, using Cu
radiation operating with 30 mA and 40 kV in the
K (1.5406 A)
2 range 1070 . A scan rate of 1 min1 was employed.
(c) Thermal analysis. Thermal behaviour of the ZnOchitosan
gel, ZnOchitosan and ZnO/gentamicinchitosan nanopowders
were followed by TG-DSC with a Netzsch TG 449C STA Jupiter.
Samples were placed in alumina crucible and heated with
10 K min1 from room temperature to 900 C, under the ow
of 20 mL min1 dried air.

62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84

88
89
90
91
92

93
94
95

96
97

Zinc acetate dihydrate, Zn(Ac)2 2H2 O, with 99.9% purity was

obtained from Merck. Absolute ethanol was used as received from
Sigma without further purication. Gentamicin solution with a
concentration of 10 mg/mL was supplied by Sigma. Chitosan (CAS
9012-76-4) was used as received from Sigma.
ZnO synthesis was done as described before (Oprea et al., 2011).
ZnOgentamicin hybrid material synthesis was performed as
described in Voicu et al. (2013).
ZnOchitosan and ZnO/gentamicinchitosan composites were
obtained starting from the relevant ZnO or ZnO/gentamicin

2.2. Experimental techniques

Please cite this article in press as: Vasile, B.S., et al., Synthesis and characterization of a novel controlled release zinc oxide/gentamicinchitosan
composite with potential applications in wounds care. Int J Pharmaceut (2013), http://dx.doi.org/10.1016/j.ijpharm.2013.11.035

98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119

120

121
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136

G Model
IJP 13750 19

ARTICLE IN PRESS
B.S. Vasile et al. / International Journal of Pharmaceutics xxx (2013) xxxxxx

Fig. 2. (a) ZnOchitosan gel (12:1) cut in shapes; (b) close view of a ZnOchitosan cube; and (c) ZnOchitosan cube after 3 months in water.

137
138
139
140
141
142
143
144
145
146
147
148
149
150
151
152
153
154
155
156
157
158
159
160
161
162
163
164
165
166
167
168
169
170
171
172
173
174
175
176

177
178
179
180
181
182
183
184
185
186
187

(d) Photoluminescence spectra. Photoluminescence spectra (PL)

were measured with a Perkin Elmer P55 spectrometer using
a Xe lamp as a UV light source at ambient temperature, in
the range 350600 nm, with all the samples dried and nely
powdered. The measurements were made with a scan speed of
200 nm min1 , slit of 10 nm, and cut-off lter of 1%. An excitation wavelength of 320 nm was used.
(e) Diffuse reectance spectra measurements were made with a
JASCO V560 spectrophotometer with solid sample accessory,
in the domain 200800 nm, with a speed of 200 nm min1 .
(f) The gentamicin release was measured photometrically by UVspectroscopy. The tests were performed in duplicate and the
results were recorded as an average. A cube with the edge of
5 mm was cut from the middle of the gel form, with the weight
of 0.1371 g (and 0.1412 g, respectively). The cube was immersed
in a baker with 10 mL aqueous phosphate buffer saline (PBS)
pH = 7.4 at 37 C. The baker was kept under magnetic stirring
at 37 C. The samples were drawn at predetermined time intervals. The solution was analyzed for gentamicin content using
UVvis spectrophotometer. Derivatization was done with ophthaldialdehyde, and absorbance measurements were done
at 332 nm wavelength as described in (Sampath and Robinson,
1990).
(g) Water elimination rate from the ZnOchitosan gel was measured gravimetrically. Cubes with same dimensions were
placed on a watch glass and weighted at determined time intervals. The results were recorded as an average.
(h) For qualitative antimicrobial assay Staphylococcus aureus ATCC
25923 and Pseudomonas aeruginosa ATCC 27853 were grown
overnight at 37 C, 200 rpm, in 5 mL Luria Broth (Oxoid).
Overnight cultures were diluted at 108 CFU/mL (0.5 Mc Farland standard density) in sterile PBS. This suspension was
used to tab inoculate 90 mm Petri dishes (Nunc) containing Mueller Hinton (MH) agar (Oxoid). 7.5 L of 10 mg/mL
plain gentamicin or ZnO/gentamicinchitosan nanosystem
was added as spots on the inoculated plates. Petri dishes
were incubated 1820 h at 37 C. After incubation time,
inhibition diameters were assessed. Experiments were performed in triplicate and repeated on at least three separate
occasions.

For quantitative assay minimum inhibitory concentrations

(MICs) of tested compounds were assessed using a binary dilution method (Grumezescu et al., 2012a). Briey, S. aureus and
P. aeruginosa overnight cultures were diluted to 103 104 CFU/mL
in sterile MH broth. Inoculated media were distributed in sterile 96 well plates and dilutions of each compound were added
in the wells. Plates were incubated for 1820 h at 37 C. After
the incubation period, MICs were established by measuring the
absorbance (Abs600), using a lab spectophotometer. Experiments
were performed in triplicate and repeated on at least three separate
occasions.

3. Results and discussion

In this study, nanocrystalline ZnO particles were prepared
through forced hydrolysis of zinc acetate under moderate conditions. The method was chosen in order to avoid contamination of
ZnO nanoparticles with ions from strong bases used currently in
the precipitation methods. We incorporated ZnO and gentamicin
coated ZnO nanoparticles into chitosan solution in order to obtain
a hybrid inorganicorganic material, with high consistency, which
can be used as bandage for wound dressing.
Unlike previous reports where the content of ZnO nanoparticles
in chitosan goes as high as few percents only (Kumar et al., 2012a,b),
we describe here a quick method to incorporate large quantities of
ZnO nanoparticles, up to 12 times higher than the chitosan content
of the solution. The obtained gel contains 15 mL water for as less
as 0.1 g solid substance, which demonstrates a 15,000% capacity or
150 times its dry weight. The gel has a high consistency, does not
ow, and can be cut in shapes with a knife. It is stable up to three
days in laboratory atmosphere and over three months if immersed
in water.
The ZnOchitosan and ZnO/gentamicinchitosan nanopowders
were investigated by X-ray diffraction. The XRD pattern for all the
ZnO based nanopowders, Fig. 3, has indicated the formation of
single-phase compound. The pattern can be indexed to a hexagonal wurtzite structure. The crystallite size of the samples can be
estimated from the Scherrer equation,
D=

0.89
cos 

Fig. 3. XRD pattern of the ZnOchitosan nanopowders 6:1 and 12:1 and
ZnO/gentamicinchitosan nanopowder 12:2:1.

Please cite this article in press as: Vasile, B.S., et al., Synthesis and characterization of a novel controlled release zinc oxide/gentamicinchitosan
composite with potential applications in wounds care. Int J Pharmaceut (2013), http://dx.doi.org/10.1016/j.ijpharm.2013.11.035

188

189
190
191
192
193
194
195
196
197
198
199
200
201
202
203
204
205
206
207
208
209
210
211
212

213

G Model
IJP 13750 19
4

ARTICLE IN PRESS
B.S. Vasile et al. / International Journal of Pharmaceutics xxx (2013) xxxxxx

Fig. 4. TEM and HRTEM images of ZnO polyhedral shaped particles and SAED pattern of planes for hexagonal ZnO [ASTM 80-0075].

214
215
216
217
218
219
220
221
222
223
224
225
226
227
228
229
230
231
232
233
234
235
236
237
238

where D is the average grain size,  is the X-ray wavelength

(0.15405 nm),  and are the diffraction angle and FWHM of
an observed peak, respectively. The strongest peak (1 0 1) at
2 = 36.24 was used to calculate the average crystallite size (D)
of ZnO particles. The estimated average crystallite size was 15 nm.
It is important to notice that the gentamicin coating and incorporation processes does not lead to a change in the structural or
chemical properties of the nanocrystalline ZnO phase. The XRD
pattern of the ZnOchitosan sample, show that the crystalline
structure of the ZnO is preserved after the incorporation of the
nanoparticles in the chitosan solution. The gentamicin coating process of ZnO nanoparticles also does not modify the crystalline
structure of ZnO.
The TEM bright eld images, Fig. 4ac, obtained on ZnO reveals
that the powder is composed from polyhedral shaped particles,
with an average particle size of approximately 15 nm. By correlating the TEM and the XRD information, as crystallite size is equal
with the nanoparticles size, we can conclude that every nanoparticle is a monocrystal. The nanopowder presents a low tendency to
form soft agglomerates.
Additional information about the structures of the nanoparticles
was found through detailed analysis with HRTEM.
The HRTEM image, Fig. 4d, shows clear lattice fringes of interplanar distances of d = 2.60 A for nanocrystalline ZnO, corresponding
to Miller indices of (0 0 2) crystallographic planes of hexagonal ZnO.

In addition, the regular succession of the atomic planes indicates

that the nanocrystallites are structurally uniform and crystalline
with almost no amorphous phase present.
The TEM images for ZnOchitosan nanopowder, Fig. 5ad, show
also tendency of ZnO nanoparticles to form agglomerates. The chitosan lm coating the ZnO nanoparticles is clearly visible as a layer
of amorphous phase, with the thickness varying between 2 and
20 nm (Fig. 5bd). The high-magnication images in bright eld
obtained for the ZnOchitosan nanoparticles shows that the chitosan lm is enveloping clusters of ZnO nanoparticles but it is also
lling the empty spaces in-between particles (Fig. 5c and d).
The HRTEM images for ZnOchitosan nanoparticles (Fig. 5e and
corf) indicate the lattice fringes of interplanar distances d = 2.47 A,
responding to Miller indices of (1 0 1), crystallographic planes of
hexagonal ZnO and d = 2.82 A (1 0 0), respectively. In addition, there
is visible the amorphous chitosan lm around the nanoparticles,
with a thickness of 2 nm.
From the selected area diffraction pattern obtained on ZnO and
ZnOchitosan nanopowders, we can state that the only crystalline
phase identied is the hexagonal form of ZnO [ASTM 80-0075].
Moreover, the SAED images of both samples conrm the Miller
indices of characteristic crystalline structures identied by XRD
(insets of Figs. 4a and 5a).
In the photoluminescence spectrum of the as-synthesized ZnO
sample (Fig. 6a) there are two regions, one at about 380400 nm

Please cite this article in press as: Vasile, B.S., et al., Synthesis and characterization of a novel controlled release zinc oxide/gentamicinchitosan
composite with potential applications in wounds care. Int J Pharmaceut (2013), http://dx.doi.org/10.1016/j.ijpharm.2013.11.035

239
240
241
242
243
244
245
246
247
248
249
250
251
252
253
254
255
256
257
258
259
260
261
262
263

G Model
IJP 13750 19

ARTICLE IN PRESS
B.S. Vasile et al. / International Journal of Pharmaceutics xxx (2013) xxxxxx

Fig. 5. TEM and HRTEM images of ZnOchitosan (12:1) dry nanopowder and SAED pattern of planes for hexagonal ZnO [ASTM 80-0075].

264
265
266
267
268
269
270
271
272
273
274
275
276
277
278
279
280
281

(near ultraviolet) and 440530 nm (blue-green), respectively. The

ZnO presents a weak UV emission band at 394 nm, corresponding
to the near band-edge emission (NBE).
The four other emission bands at 446, 456, 482, and 514528 nm
in the blue-green range are defect-related emissions, indicating a
high concentration of surface defects, as expected for polyhedral
shape particles (Oprea et al., 2013). The 482 nm band is relatively
common for semiconductor oxides like MIn2 O4 (M = Ca, Sr, Ba) or
SnO2 (Gingasu et al., 2011) conrming that it is caused by the oxygen related defects (Sabri et al., 2012).
The photoluminescence spectra of gentamicin coated ZnO (Fig.
6b) presents same transitions as ZnO in the green region (482 nm
peak and 514528 nm broad band) but with higher intensities. The
band from 456 nm in ZnO is shifted to 464 nm in ZnOgentamicin
nanopowder and the NBE increases in intensity and appears shifted
at 402 nm. The enhanced luminescence of the ZnOgentamicin
hybrid can be attributed to the presence of the organic substance
and (OH) groups on the surface of the ZnO (Jiang et al., 2007; Peng

and Wang, 2010). As the gentamicin forms an organic shell around

ZnO nanoparticles, according to literature, the enhancement of
photoluminescence emission is caused by the removal of electron
capture centres on the surface of nanocrystals or the removal of
nonradiative decay channels (Liu et al., 2011).
In the case of ZnO/gentamicinchitosan nanopowder (Fig. 6c)
the emission band from 402 nm is diminished in intensity and
shifted to 400 nm. In the blue region of the emission spectra,
the intensity of the bands is increased and more peaks can be
noticed. As the emission spectrum is different by large extent
from that of the ZnOchitosan nanopowder, but more similar
with that of ZnO/gentamicin, this can be consider as an indication
that the structure of the coating layers is preserved in the order
ZnO/gentamicinchitosan.
The emission spectra of ZnOchitosan nanopowder (Fig. 7d)
consists of only one, asymmetric, very intense emission peak, with
the maximum at 381 nm, assigned to the NBE. The intensity of
this emission band is eight times higher than the one recorded

Please cite this article in press as: Vasile, B.S., et al., Synthesis and characterization of a novel controlled release zinc oxide/gentamicinchitosan
composite with potential applications in wounds care. Int J Pharmaceut (2013), http://dx.doi.org/10.1016/j.ijpharm.2013.11.035

282
283
284
285
286
287
288
289
290
291
292
293
294
295
296
297
298
299

G Model
IJP 13750 19
6

ARTICLE IN PRESS
B.S. Vasile et al. / International Journal of Pharmaceutics xxx (2013) xxxxxx

Fig. 6. Photoluminescence spectra of (a) ZnO nanopowder; (b) ZnO/gentamicin

nanopowder; and (c) ZnO/gentamicinchitosan nanopowder (12:2:1).

300
301
302
303
304
305
306
307
308
309
310
311
312
313
314
315
316
317
318
319
320
321
322

for ZnO/gentamicin and over thirty times higher than the one
corresponding to the bare ZnO nanoparticles. Because surface optical emission efciency can increase over tenfold as roughness
decreases to unit cell dimensions (Doutt et al., 2009), we can
attribute this high intensity of NBE to the smoothing of the nanoparticles surface during incorporation into the chitosan solution.
Such high intensity emission band can be further used in other
medical application, such as photodiagnosis or biosensing (Zhao
et al., 2013).
The electronic spectra recorded for the uncoated and gentamicin coated ZnO powder is presented in Fig. 8. The calculated band
gap for ZnO is 3.36 eV, in good agreement with the literature. The
absorbance of the ZnOchitosan nanopowder is slightly higher than
that of uncoated ZnO, in the visible region, the layer of chitosan
contributing to the light absorption. For ZnO/gentamicinchitosan
the absorbance in visible region is further increased as the organic
layer contains two components. As the spectrum is recorded in
reectance mode, the photons that are not absorbed by ZnO must
travel twice through the gentamicin and chitosan layer, yielding a
higher absorbance. The maximum absorbance for all the samples
is at 363 nm.
thermal
analysis
of
ZnOchitosan
and
The
ZnO/gentamicinchitosan nanopowders (Fig. 9) are similar up to

Fig. 7. Comparative photoluminescence spectra of ZnOchitosan nanopowder

(12:1) vs. ZnO, ZnO/gentamicin and ZnO/gentamicinchitosan (12:2:1).

Fig. 8. Diffuse reectance spectra of

ZnO/gentamicinchitosan nanopowders.

(a)

ZnO;

(b)

ZnOchitosan);

(c)

200 C. The samples present a mass loss of 2.5%, corresponding to

the residual water, still trapped in the ZnOchitosan network. The
ZnOchitosan exhibit a slightly better thermal stability than the
ZnO/gentamicinchitosan, most probably due to the direct bonds
formed with ZnO nanoparticles.
ZnOchitosan powder present a single decomposition step
between 200 and 500 C, accompanied by a weak exothermic effect
on the DSC curve. This step corresponds to the oxidation of the
organic part. The residual mass, 88%, corresponds to the ZnO
(close to the ration of 12:1 used in synthesis).
The ZnO/gentamicinchitosan presents an additional mass loss
in the 200500 C interval, as beside oxidation of the chitosan,
there is a partial decomposition of the gentamicin as well. The
nal oxidation of gentamicin takes place between 500 and 650 C,
accompanied by a strong exothermic process on the DSC curve.
The residual mass formed by ZnO represents 73%, corresponding to the synthesis ratio of 12:2:1 (ZnO:gentamicin:chitosan). The
analysis is in good agreement with previously reported results for
thermal behaviour of gentamicin (Simchua et al., 2011).
The gentamicin release rate was monitored to reveal if the entire
antibiotic amount is made available in a short period of time or the
ZnOchitosan gel can sustain a prolonged release.
We have found in a previous study (Voicu et al., 2013), that gentamicin was loosely adsorbed on the surface of the individual ZnO
particles, more than 80% being released in the rst 10 min. This
is not the case with the ZnO/gentamicinchitosan gel. Although
the release curve (Fig. 10) present an initial burst, 16% in rst
10 min, the release rate then slows down, 40% of gentamicin being
released in rst hour. A second increase of the release rate can be
observed between 2 and 3 h when another 25% of gentamicin is
released from the gel. After 8 h 91% of the gentamicin amount is
released.
The most probably structure adopted by the ZnOchitosan gel is
represented in Fig. 11. The ZnO nanoparticles are interacting with
amino groups to closely wrap around the chitosan chains. As a
result, the structure of the gel resembles that of an egg box, with
channels generated by the chitosan chains wrapped around zinc
oxide nanoparticles. The structure of the gel contains additional
cavities in which water is trapped. There are two types of cavities,
A and B (Fig. 11b) in the gel structure. First one (A type) is represented by the spaces generated by the channel network. Second
one (B type) is represented by the channels spaces, in which ZnO
nanoparticles are placed. Water is to be found in both types of cavities. As the release of gentamicin starts as soon ZnO/gentamicin

Please cite this article in press as: Vasile, B.S., et al., Synthesis and characterization of a novel controlled release zinc oxide/gentamicinchitosan
composite with potential applications in wounds care. Int J Pharmaceut (2013), http://dx.doi.org/10.1016/j.ijpharm.2013.11.035

323
324
325
326
327
328
329
330
331
332
333
334
335
336
337
338
339
340
341
342
343
344
345
346
347
348
349
350
351
352
353
354
355
356
357
358
359
360
361
362
363
364
365
366

G Model
IJP 13750 19

ARTICLE IN PRESS
B.S. Vasile et al. / International Journal of Pharmaceutics xxx (2013) xxxxxx

Fig. 9. Thermal analysis of (a) ZnOchitosan; and (b) ZnO/gentamicinchitosan nanopowders.

367
368
369
370
371
372
373
374
375
376
377
378
379
380
381
382
383
384
385
386
387
388
389
390

nanopowder is suspended in water, is fair to assume that at the

time gel is formed, part of gentamicin is already solubilized in the
gel-trapped water, in A type cavities. With this model in mind, the
gentamicin release mechanism can be explained as follow.
The initial fast release shows that the release kinetic is dominated by adsorption/desorption and by diffusion out of the gel.
Normally all the gentamicin is loosely bound to the surface of the
ZnO nanoparticles and will diffuse out into the water phase, as gentamicin is highly hydrophilic. In the rst phase only the gentamicin
that is already dissolved in the water in A type cavities can diffuse
out of the gel. The gentamicin that is trapped inside the channels
(B type cavities) will slowly diffuse in to the network spaces (A
type cavities) and then out of the gel. As the concentration of gentamicin in the channels decrease, the ZnO bounded gentamicin is
released to re-establish the equilibrium. In this way, even if we have
a continuous release of gentamicin, the rate is variable because of
multiple processes that take place until the nal release from the
gel.
To verify the model, and in order to determine the stability of the
ZnOchitosan gel, we have measured the amount of water loosed
over time, until the gel dries out to form a lm (Fig. 12).
It can be observed that after the initial mass loss, the gel has a
relative stability over a period of 2 days in which water is slowly
removed. Following the structure collapse after 2 days, the water

is quickly removed, and a lm is obtained. This conrms the model

proposed previously, as the initial mass loss can be attributed to the
surface network water, which is the rst to exit the gel structure
(from A type cavities). Most probably, after the water is expelled
from the surface, the chitosan network shrinks so any further water
loss is occurring much slower. This can explain the relative stability
of the gel.

Fig. 10. The gentamicin release from the ZnO/gentamicin nanopowder and
ZnO/gentamicinchitosan gel.

Fig. 11. The proposed models of (a) ZnO nanoparticleschitosan interaction; and
(b) ZnOchitosan gel structure.

Please cite this article in press as: Vasile, B.S., et al., Synthesis and characterization of a novel controlled release zinc oxide/gentamicinchitosan
composite with potential applications in wounds care. Int J Pharmaceut (2013), http://dx.doi.org/10.1016/j.ijpharm.2013.11.035

391
392
393
394
395
396
397

G Model
IJP 13750 19
8

ARTICLE IN PRESS
B.S. Vasile et al. / International Journal of Pharmaceutics xxx (2013) xxxxxx

Fig. 12. Stability of ZnOchitosan gel in laboratory atmosphere.

398
399
400
401
402
403
404
405
406
407
408
409
410
411
412
413
414
415
416
417
418
419
420

If the gel is kept in water, then there will be no chitosan network

modications and the gel is stable for more than 3 months (Fig. 2c).
The antimicrobial assay demonstrates that the prepared material has a great antimicrobial activity in both planktonic and
surface-attached conditions. Results obtained after the qualitative
assay demonstrated that growth inhibition diameters are signicantly enhanced by the addition of active antibiotic-nanosystem.
ZnO/gentamicinchitosan enhanced the growth inhibition zone
from 11 mm to 17 mm for S. aureus and from 13 mm to 17 mm for
P. aeruginosa, as compared with gentamicin control (Fig. 13).
The MIC assay revealed that ZnO/gentamicinchitosan active
nanosystem inhibits both S. aureus and P. aeruginosa growth at a
much lower concentration comparing to gentamicin control. For
S. aureus, MIC value was reduced from 0.48 g/mL to 0.12 g/mL,
while for P. aeruginosa MIC value was reduced from 1.95 g/mL to
0.97 g/mL (Fig. 14).
The obtained results suggest that this active nanosystem based
on ZnO/antibioticchitosan nanostructures has the potential to
be efciently used for biomedical applications, exhibiting great
antimicrobial properties. This nanosystem signicantly enhanced
the antibacterial activity of gentamicin against two highly versatile
opportunistic pathogens S. aureus and P. aeruginosa, probably by
acting as an efcient drug delivery and controlled release system.

Fig. 14. Graphic representation of MICs obtained after growing S. aureus and
P. aeruginosa in the presence of diferent concentrations of gentamicin and
ZnO/gentamicinchitosan.

4. Conclusions
In this paper we describe an easy method to incorporate ZnO
nanoparticles into a chitosan solution at a weight ratio up to 12:1.
The obtained gel has proved a high water content (99.33% weight),
but also a high consistency, maintaining its shape after cutting.
Furthermore, this product is stable up to three days in laboratory atmosphere, and over three months if immersed in water.
The incorporation of gentamicin loaded ZnO nanoparticles in chitosan solution yields a three-component gel, with a slow release
rate of the drug. As all three components have antibacterial activity, a synergic action was expected. The results proved a four-fold
MIC reduction for S. aureus and a two-fold reduction of MIC for P.
aeruginosa. The gel can be used in topic applications that requires
a large spectrum antibacterial activity, namely as a bandage for
wound dressing. The high water content help in maintaining a
moist environment at the wound interface, providing a cooling
sensation and soothing effect, much like aloe vera base gel, while
slowly releasing the antibiotic. Most important, the system is fully
scalable to any other soluble drug, as the entire solution remains
trapped in the ZnOchitosan gel.
In addition, for the ZnOchitosan nanopowder we report here
an unusual high intensity for the UV emission band, property that
can be further used in various medical elds like photodiagnosis or
biosensing.
Acknowledgment
Authors recognize nancial support from the European Social
Fund through POSDRU/89/1.5/S/54785 project: Postdoctoral Program for Advanced Research in the Field of Nanomaterials.
References

Fig. 13. Graphic representation of inhibition diameters (mm) of S. aureus and P.

aeruginosa grown in the presence of gentamicin and ZnO/gentamicinchitosan.

Andronescu, E., Grumezescu, A.M., Ficai, A., Gheorghe, I., Chiriuc, M., Mihaiescu,
D.E., Lazar, V., 2012. In vitro efcacy of antibiotic magnetic dextran microspheres
complexes against Staphylococcus aureus and Pseudomonas aeruginosa strains.
Biointerface Res. Appl. Chem. 2, 332338.

Please cite this article in press as: Vasile, B.S., et al., Synthesis and characterization of a novel controlled release zinc oxide/gentamicinchitosan
composite with potential applications in wounds care. Int J Pharmaceut (2013), http://dx.doi.org/10.1016/j.ijpharm.2013.11.035

421

422
423
424
425
426
427
428
429
430
431
432
433
434
435
436
437
438
439
440
441
442
443
444

445

446
447
448

449

450
451
452
453

G Model
IJP 13750 19

ARTICLE IN PRESS
B.S. Vasile et al. / International Journal of Pharmaceutics xxx (2013) xxxxxx

454
455
456
457
458
459
460
461
462
Q2
463
464
465
466
467
468
469
470
471
472
473
474
475
476
477
478
479
480
481
482
483
484
485
486
487
488
489
490
491
492
493
494
495
496
497
498
499
500
501
502
503
504
505
506
507
508
509
510
511
512
513
514
515
516
517
518

Archana, D., Dutta, J., Dutta, P.K., 2013. Evaluation of chitosan nano dressing for
wound healing: Characterization, in vitro and in vivo studies. Int. J. Biol. Macromol. 57, 193203.
Builders, P.F., Kabele-Toge, B., Builders, M., Chindo, B.A., Anwunobi, P.A., Isimi, Y.C.,
2013. Wound healing potential of formulated extract from Hibiscus sabdariffa
calyx. Indian J. Pharm. Sci. 75, 4552.
Casettari, L., Vllasaliu, D., Lam, J.K.W., Soliman, M., Illum, L., 2012. Biomedical applications of amino acid-modied chitosans: a review. Biomaterials 33, 75657583.
Doutt, D., Mosbacker, H.L., Cantwell, G., Zhang, J., Song, J.J., Brillson, L.J., 2009. Impact
of near-surface defects and morphology on ZnO luminescence. Appl. Phys. Lett.
94.
Eidi, H., Joubert, O., Nemos, C., Grandemange, S., Mograbi, B., Foliguet, B., Tournebize,
J., Maincent, P., Le Faou, A., Aboukhamis, I., Rihn, B.H., 2012. Drug delivery by
polymeric nanoparticles induces autophagy in macrophages. Int. J. Pharm. 422,
495503.
El Shafei, A., Abou-Okeil, A., 2011. ZnO/carboxymethyl chitosan bionano-composite
to impart antibacterial and UV protection for cotton fabric. Carbohydr. Polym.
83, 920925.
Gingasu, D., Oprea, O., Mindru, I., Culita, D.C., Patron, L., 2011. Alkali earth metal
indates synthesized by precursor method. Digest J. Nanomater. Biostruct. 6,
12151226.
Grumezescu, A.M., Andronescu, E., Ficai, A., Bleotu, C., Chiriuc, M.C., 2012a. Chitin
based biomaterial for antimicrobial therapy: fabrication, characterization and
in vitro prole based interaction with eukaryotic and prokaryotic cells. Biointerface Res. Appl. Chem. 2, 438445.
Grumezescu, A.M., Andronescu, E., Ficai, A., Bleotu, C., Mihaiescu, D.E., Chiriuc, M.C.,
2012b. Synthesis, characterization and in vitro assessment of the magnetic chitosancarboxymethylcellulose biocomposite interactions with the prokaryotic
and eukaryotic cells. Int. J. Pharm. 436, 771777.
Grumezescu, A.M., Andronescu, E., Ficai, A., Mihaiescu, D.E., Vasile, B.S., Bleotu,
C., 2012c. Synthesis, characterization and biological evaluation of a Fe3 O4 /C12
core/shell nanosystem. Lett. Appl. NanoBioSci. 1, 3135.
Grumezescu, A.M., Holban, A.M., Andronescu, E., Ficai, A., Bleotu, C., Chiriuc, M.C.,
2012d. Water dispersible metal oxide nanobiocomposite as a potentiator of the
antimicrobial activity of kanamycin. Lett. Appl. NanoBioSci. 1, 7782.
Grumezescu, A.M., Mihaiescu, D.E., Tamas, D., 2012e. Hybrid materials for drug delivery of rifampicin: evaluation of release prole. Biointerface Res. Appl. Chem. 1,
229235.
Jiang, D.X., Cao, L.X., Su, G., Qu, H., Sun, D., 2007. Luminescence enhancement of
Mn doped ZnS nanocrystals passivated with zinc hydroxide. Appl. Surf. Sci. 253,
93309335.
Kumar, P.T.S., Lakshmanan, V.K., Anilkumar, T.V., Ramya, C., Reshmi, P., Unnikrishnan, A.G., Nair, S.V., Jayakumar, R., 2012a. Flexible and microporous chitosan
hydrogel/nano ZnO composite bandages for wound dressing: in vitro and in vivo
evaluation. ACS Appl. Mater. Interfaces 4, 26182629.
Kumar, P.T.S., Lakshmanan, V.K., Biswas, R., Nair, S.V., Jayakumar, R., 2012b. Synthesis
and biological evaluation of chitin hydrogel/nano ZnO composite bandage as
antibacterial wound dressing. J. Biomed. Nanotechnol. 8, 891900.
Liu, Q., Ren, W.T., Zhang, Y., Zhang, Y.X., 2011. Hydrogenated carboxylated nitrite
rubber/modied zinc carbonate basic composites with photoluminescence
properties. Eur. Polym. J. 47, 11351141.
Liu, Y., Kim, H.I., 2012. Characterization and antibacterial properties of genipincrosslinked chitosan/poly(ethylene glycol)/ZnO/Ag nanocomposites. Carbohydr. Polym. 89, 111116.
Manjunatha, N., Vasanti, S., Rajesh, N., Uma, N., 2010. Formulation and evaluation of
biopolymer based microspheres for nasal drug delivery. Int. J. PharmTech Res.
2, 856862.
Mihaiescu, D.E., Grumezescu, A.M., Balaure, P.C., Mogosanu, D.E., Traistaru, V., 2011.
Magnetic scaffold for drug targeting: evaluation of cephalosporins controlled
release prole. Biointerface Res. Appl. Chem. 1, 191195.
Mihaiescu, D.E., Horja, M., Gheorghe, I., Ficai, A., Grumezescu, A.M., Bleotu, C., Chiriuc, C.M., 2012. Water soluble magnetite nanoparticles for antimicrobial drugs
delivery. Lett. Appl. NanoBioSci. 1, 4549.
Oprea, O., Andronescu, E., Vasile, B.S., Voicu, G., Covaliu, C., 2011. Synthesis and
characterization of ZnO nanopowder by non-basic route. Digest J. Nanomater.
Biostruct. 6, 13931401.

Oprea, O., Vasile, O.R., Voicu, G., Andronescu, E., 2013. The inuence of the thermal
treatment on luminescence properties of ZnO. Digest J. Nanomater. Biostruct. 8,
747756.
Peng, L.L., Wang, Y.H., 2010. Effects of the template composition and coating on the
photoluminescence properties of ZnS:Mn nanoparticles. Nanoscale Res. Lett. 5,
839845.
Perelshtein, I., Ruderman, E., Perkas, N., Tzanov, T., Beddow, J., Joyce, E., Mason, T.J.,
Blanes, M., Molla, K., Patlolla, A., Frenkel, A.I., Gedanken, A., 2013. Chitosan and
chitosanZnO-based complex nanoparticles: formation, characterization, and
antibacterial activity. J. Mater. Chem. B 1, 19681976.
Ribeiro, M.P., Morgado, P.I., Miguel, S.P., Coutinho, P., Correia, I.J., 2013. Dextranbased hydrogel containing chitosan microparticles loaded with growth factors
to be used in wound healing. Mater. Sci. Eng. C: Mater. Biol. Appl. 33, 29582966.
Sabri, N.S., Yahya, A.K., Talari, M.K., 2012. Emission properties of Mn doped
ZnO nanoparticles prepared by mechanochemical processing. J. Lumin. 132,
17351739.
Sampath, S.S., Robinson, D.H., 1990. Comparison of new and existing spectrophotometric methods for the analysis of tobramycin and other aminoglycosides. J.
Pharm. Sci. 79, 428431.
Saviuc, C., Grumezescu, A.M., Holban, A., Bleotu, C., Chiriuc, C., Balaure, P., Lazar,
V., 2011. Phenotypical studies of raw and nanosystem embedded Eugenia carryophyllata buds essential oil antibacterial activity on Pseudomonas aeruginosa
and Staphylococcus aureus strains. Biointerface Res. Appl. Chem. 1, 111118.
Selvam, S., Rajiv Gandhi, R., Suresh, J., Gowri, S., Ravikumar, S., Sundrarajan, M.,
2012. Antibacterial effect of novel synthesized sulfated cyclodextrin crosslinked
cotton fabric and its improved antibacterial activities with ZnO, TiO2 and Ag
nanoparticles coating. Int. J. Pharm. 434, 366374.
Shalumon, K.T., Anulekha, K.H., Nair, S.V., Nair, S.V., Chennazhi, K.P., Jayakumar, R.,
2011. Sodium alginate/poly(vinyl alcohol)/nano ZnO composite nanobers for
antibacterial wound dressings. Int. J. Biol. Macromol. 49, 247254.
Simchua, W., Narkkong, N.A., Baimark, Y., 2011. Silk broin nanospheres for controlled gentamicin sulfate delivery. Res. J. Nanosci. Nanotechnol. 1, 3441.
Sogias, I.A., Williams, A.C., Khutoryanskiy, V.V., 2012. Chitosan-based mucoadhesive
tablets for oral delivery of ibuprofen. Int. J. Pharm. 436, 602610.
Song, Y., Du, X., Li, T.M., Zhu, Y.J., Li, M., 2013. Phenotypic and molecular characterization of Staphylococcus aureus recovered from different clinical specimens of
inpatients at a teaching hospital in Shanghai between 2005 and 2010. J. Med.
Microbiol. 62, 274282.
Subhasree, R.S., Selvakumar, D., Kumar, N.S., 2012. Hydrothermal mediated synthesis of ZnO nanorods and their antibacterial properties. Lett. Appl. NanoBioSci. 1,
27.
Sun, F.J., Zhang, X.B., Fang, Y.D., Chen, J.H., Xing, H.Y., Shi, H.Q., Feng, W., Xia, P.Y.,
2012. Spectrum and drug resistance of pathogens from patients with burns.
Burns 38, 11241130.
Vaja, F., Comanescu, C., Oprea, O., Ficai, D., Guran, C., 2012. Effects of ZnO nanoparticles on the wet scrub resistance and photocatalytic properties of acrylic coatings.
Rev. Chim. 63, 722726.
Vaja, F., Ficai, D., Ficai, A., Oprea, O., Guran, C., 2013. Multifunctional advanced coatings based on ZnO/M obtained by nanocasting method. J. Optoelectron. Adv.
Mater. 15, 107113.
Vlad, S., Tanase, C., Macocinschi, D., Ciobanu, C., Balaes, T., Filip, D., Gostin, I.N., Gradinaru, L.M., 2012. Antifungal behaviour of polyurethane membranes with zinc
oxide nanoparticles. Digest J. Nanomater. Biostruct. 7, 5158.
Voicu, G., Oprea, O., Vasile, B.S., Andronescu, E., 2013. Antibacterial activity of zinc
oxidegentamicin hybrid material. Digest J. Nanomater. Biostruct. 8, 11911203.
Wijekoon, A., Fountas-Davis, N., Leipzig, N.D., 2013. Fluorinated methacrylamide
chitosan hydrogel systems as adaptable oxygen carriers for wound healing. Acta
Biomater. 9, 56535664.
Zabielinski, M., McLeod, M.P., Aber, C., Izakovic, J., Schachner, L.A., 2013. Trends
and antibiotic susceptibility patterns of methicillin-resistant and methicillinsensitive Staphylococcus aureus in an outpatient dermatology facility. JAMA
Dermatol. 149, 427432.
Zhao, M.G., Huang, J.Y., Zhou, Y., Chen, Q., Pan, X.H., He, H.P., Ye, Z.Z., 2013. A single
mesoporous ZnO/Chitosan hybrid nanostructure for a novel free nanoprobe type
biosensor. Biosens. Bioelectron. 43, 226230.

Please cite this article in press as: Vasile, B.S., et al., Synthesis and characterization of a novel controlled release zinc oxide/gentamicinchitosan
composite with potential applications in wounds care. Int J Pharmaceut (2013), http://dx.doi.org/10.1016/j.ijpharm.2013.11.035

519
520
521
522
523
524
525
526
527
528
529
530
531
532
533
534
535
536
537
538
539
540
541
542
543
544
545
546
547
548
549
550
551
552
553
554
555
556
557
558
559
560
561
562
563
564
565
566
567
568
569
570
571
572
573
574
575
576
577
578
579
580
581
582
583