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Chitosan nanoparticles for the delivery of a new compound active against multidrug-resistant Staphylococcus aureus
Journal Pre-proof Chitosan nanoparticles for the delivery of a new compound active against multidrugresistant Staphylococcus aureus Laura Freitas de Andrade, Alexsandra Conceição Apolinário, Carlota de Oliveira Rangel-Yagui, Marco Antônio Stephano, Leoberto Costa Tavares PII:
S1773-2247(19)31087-1
DOI:
https://doi.org/10.1016/j.jddst.2019.101363
Reference:
JDDST 101363
To appear in:
Journal of Drug Delivery Science and Technology
Received Date: 31 July 2019 Revised Date:
21 October 2019
Accepted Date: 28 October 2019
Please cite this article as: L.F. de Andrade, Alexsandra.Conceiçã. Apolinário, C. de Oliveira RangelYagui, Marco.Antô. Stephano, L.C. Tavares, Chitosan nanoparticles for the delivery of a new compound active against multidrug-resistant Staphylococcus aureus, Journal of Drug Delivery Science and Technology (2019), doi: https://doi.org/10.1016/j.jddst.2019.101363. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2019 Published by Elsevier B.V.
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Chitosan nanoparticles for the delivery of a new compound active against multidrug-resistant Staphylococcus aureus
3 4 5 6
Laura Freitas de Andrade, Alexsandra Conceição Apolinário, Carlota de Oliveira Rangel-Yagui, Marco Antônio Stephano, Leoberto Costa Tavares*
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Department of Biochemical and Pharmaceutical Technology, Faculty of Pharmaceutical Sciences, University of Sao Paulo, São Paulo, Brazil.
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**Correspondence Author:
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[email protected]
11
University of Sao Paulo
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Av. Prof. Lineu Prestes, 580
13
05508-000 - São Paulo, SP, Brazil
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+ 55 11 30912385
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Abstract: Chitosan is a biopolymer with antimicrobial, analgesic activity, tissue regenerator properties and biofilm protection. The N’-((5-nitrofuran-2-yl)methylen)-2-benzhydrazide, a novel active compound against multidrug-resistant Staphylococcus aureus (5-NFB), was incorporated in Polysorbate 20 micelles and further loaded in chitosan nanoparticles (Ch-5-NFB-NP), prepared by ionic gelation varying NaCl concentration. The nanoparticles were characterized by Dynamic Light Scattering (DLS) to determine size, polydispersity index (PDI) and ζ-potential. Encapsulation Efficiency (EE%) was determined by indirect method and morphology by scanning electronic microscopy (SEM). Antimicrobial activity tests against Staphylococcus aureus strains ATCC 29213, hVISA and ORSA were performed with 5-NFB, Ch-5-NFB-NP and empty chitosan nanoparticles (Ch-NP) using colorimetric and microdilution methods by minimal inhibitory concentration (MIC). In the optimal experiment, the Ch-5-NFB-NP were obtained with average diameter of 321 nm, PDI of 0.18, ζ-P of +37 mV; EE% of 44% and the morphology by SEM showed spherical and regular shaped nanoparticles. The best results for bacterial growth inhibition against all strains tested were observed for Ch-5-NFB-NP. The nanoparticles were lyophilized with different lyoprotectants and the best freeze-dried samples were obtained with lactose and saccharose, keeping the Ch-5-NFB-NP characteristics. Therefore, owing to the antibacterial activity, tissue regenerator property and protective biofilm effect, the Ch-5-NFB-NP are a promising alternative to treat multidrug-resistant infections, especially in burned patients.
34 35 36
Keywords: Nifuroxazide; Chitosan; Nanoparticles; Drug delivery; Multidrug-resistant bacteria; Lyophilization.
2
37
1. INTRODUCTION
38
Antibiotic resistance is one of the main concerns in global health and bacterial resistant
39
strains results in many deaths in hospitals [1]. Infections by multidrug-resistant bacteria is
40
responsible for 700 thousand deaths per year worldwide and this number may increase giving the
41
indiscriminate use of antibiotics in therapeutics and farming, as well as incorrect discharge of
42
these substances[1]. Among the most threatening bacteria, one can highlight penicillin resistant
43
Staphylococcus epidermidis and Staphylococcus aureus, in particular the following strains:
44
methicilin resistant S. aureus (MRSA), oxacillin resistant S. aureus (ORSA), vancomycin
45
intermediate S. aureus (VISA) and heterogeneous vancomycin intermediate S. aureus (hVISA).
46
Multidrug-resistant strains of anaerobic and facultative aerobic bacteria as S. aureus usually
47
infect burns and cutaneous lesions of large extension, leading to high rates of sepsis in patients
48
and, consequently, high morbidity and mortality [2]. Within this context, novel molecules and/or
49
pharmaceutical formulations with significative activity against these resistant strains are of great
50
interest.
51
Our group has recently synthesized a 5-nitro-2-heterocyclic compound analog to
52
nifuroxazide
(Fig.
1)
that
presented
significant
activity
against
multidrug-resistant
53
microorganisms as S. aureus, S. epidermidis and C. albicans [3]. Although the mechanism of
54
action of this class is not fully elucidated, the bactericidal effect seems to be related to the
55
reduction of the nitro group (-NO2) leading to the generation of free radicals that, in turn,
56
interfere with the cellular metabolism, as DNA and RNA structures and mitochondrial
57
respiration. The nitro-group is more selective to bacterial, fungal and protozoan cells than human
58
cells and the major reduction potential of nitro group is directly linked to increased cellular death
59
[4].
60
61 62 63
Figure 1- Chemical structure of nifuroxazide and its 5-NFB analog: N’-((5-nitrofuran-2-
64
yl)methylene)-2-benzhydrazide.
65
3
66
The nifuroxazide analog N’-((5-nitrofuran-2-yl)methylene)-2-benzhydrazide (5-NFB) can be
67
considered a promising alternative to treat infections by multidrug-resistant microorganisms, it
68
was one of the most potent among the nitro-compound series previously studied [3]. However,
69
the high lipophilicity hinders further evaluation and potential application of this molecule. In this
70
sense, incorporation into nanostructures can be an alternative for the delivery of 5-NFB by
71
promoting higher solubility and improving bioavailability. Additionally, topical administration of
72
nanostructured antimicrobials is advantageous for effective treatment of local disease, such as
73
burned areas, due to their inherent ability to circumvent systemic cytotoxicity and ease of rapid
74
delivery at the site of infection [5,6].
75
Nanostructures based on chitosan biopolymer have been widely studied as drug delivery
76
systems owing to its biocompatibility and low toxicity [7]. Chitosan is an oligomer formed by D-
77
glucosamine and D-glucoacetamide units linked by glucosides β-(1 > 4) bonds (Fig. 2). It is
78
obtained by chitin deacetylation, which provides the polycationic character of chitosan by amines
79
protonation in acid environment [8]. This polymer is able to bind polyanions as sodium
80
tripolyphosphate (TPP), forming hydrogels that can be employed as biofilms, nanoparticles and
81
microparticles [8,9].
82
One interesting point of using chitosan as a biomaterial for nanostructures preparation is that
83
it has analgesic, healing and antibacterial pharmacological activities. The antimicrobial activity of
84
nicin and natamycin incorporated into chitosan nanoparticles, for example, was proved against
85
Salmonella enterica and Penicillium chrysogenum, with 50% and 12.5% of reduction in MIC
86
compared to free-nicin and free-natamycin [11]. Additionally, it presents mucoadhesive
87
proprieties with biofilm formation. All these properties have been encouraging the investigation
88
of chitosan as scaffold for tissue regeneration. The antibacterial activity of chitosan films and
89
chitosan nanoparticles is related to the positive charges of chitosan that bind to the negative
90
charges in bacteria, but the mechanisms of action is not fully known [10]. Chitosan nanoparticles
91
have also been widely studied to increase apparent solubility and bioavailability of drugs. The
92
mucoadhesive property and biofilm formation of chitosan enable higher and prolonged contact to
93
the administration route [12,13], what is of great interest for skin infections such as the ones
94
caused by S. aureus strains.
95
In this paper, we developed chitosan nanoparticles loaded with N’-((5-nitrofuran-2-
96
yl)methylene)-2-benzhydrazide (Ch-5-NFB-NP) by ionic gelation method aiming an stable
4
97
delivery systems. The 5-NFB-loaded chitosan NP were proved to be active in vitro against S.
98
aureus ATCC 29213, hVISA and ORSA strains. Additionally, we investigated the lyophilization
99
process for the nanoparticles developed using glycine, lactose and saccharose as lyoprotectants.
100 101
2. MATERIALS AND METHODS
102
2.1. Materials
103
Chitosan (low molecular weight of 50 – 190 kDa and deacetylaytion degree of 75 – 85%),
104
sodium tripolyphosphate (85% of purity), Mueller Hington broth and bromocresol purple were
105
purchased from Sigma Aldrich (St. Louis, MO, USA). N’-((5-nitrofuran-2-yl)methylen)-2-
106
benzhydrazyde compound (5-NFB) was previously synthesized by our group and duly purified by
107
DMF recrystallization and identified by NMR H1 and NMR C13 (Zorzi et al., 2014). All other
108
reagents were purchased from LabSynth (Diadema, SP, BRA).
109
2.2. Preparation of empty chitosan nanoparticles (Ch-NP) and chitosan/5-NFB nanoparticles
110
(Ch-5-NFB-NP)
111
The Ch-NP and Ch-5-NFB-NP preparation was adapted from a previously described method
112
[11]. Briefly, chitosan (2 mg/mL) was dissolved in acetic acid 1% (pH 5.2); ethanol (10%) and
113
Polysorbate 20 (1.4 mM) were added in the solution. Ch-NP were produced by the dropwise
114
addition of a TPP solution (1 mg/mL) up to 3:1 chitosan/TPP ratio, under magnetic stirring at
115
room temperature during 1 h. For the Ch-5-NFB-NP, a solution of 5-NFB in ethanol (1 mg/mL)
116
was slowly added to the chitosan/ethanol/Polysorbate 20 solution under magnetic stirring before
117
TPP addition. The effect of ionic strength on the Ch-5-NFB-NP colloidal stability was
118
investigated by varying the NaCl concentration (0.75 and 155 mM) in the chitosan and TPP
119
solutions.
120
NPs were purified by centrifugation at 13,000 x g in 10 µL of pure glycerol as support into
121
Eppendorf flasks for 15 minutes. The supernatant was used for indirect determination of the drug
122
encapsulation efficiency (EE%) and the precipitated nanoparticles were resuspended with
123
ultrapure water completing the previous volume.
124
2.3. Nanoparticles Characterization
5
125
The NPs samples were prepared as explained above and they were analyzed by photon
126
correlation spectroscopy (Dynamic Light Scattering) to determine average size and zeta potential
127
both using the Zetasizer Nano ZS (Malvern Instruments Ltd, Worcestershire,UK). The size and
128
polydispersity index were measured in an angle of 175º and 532 nm laser, using glass cuvettes.
129
Stokes Einstein equation provided the hydrodynamic diameter from diffusion coefficient values
130
(Equation 1). In addition, particle size distribution was provided by cumulant analysis method.
131
The mobility was then related to the ζ-potential using the Henry equation (Equation 2). All
132
measurements were performed at least three times.
133 =
134
(Equation 1)
135
in which: kB = Boltzmann constant (1.38064852 × 10 − 23 J/K), T = temperature, η = absolute
136
viscosity of the PBS and Rh = hydrodynamic radius.
137 UE =
138
(
)
(Equation 2)
139
in which: UE = electrophoretic mobility, z = zeta potential, ε = dielectric constant, η = viscosity
140
and f(κa) = Henry’s function.
141 142
2.4. Encapsulation efficiency
143
The percentage of encapsulation efficiency (EE%) was calculated indirectly from the
144
difference between the initial total amount of 5-NFB added to the system and the amount of 5-
145
NFB in the supernatant after centrifugation (free compound), as presented in Eq. 3. The 5-NFB
146
concentration in supernatant was measured by UV spectrophotometry at 370 nm, in a UV/VIS
147
spectrophotometry (Spectra Max Plus 384– Molecular Devices).
148 149
EE% = Total amount of 5-NFB – Supernatant amount of 5-NFB x 100
150 151 152
Total amount of 5-NFB 2.6. Thermal analyses
(Equation 3)
6
153
Thermal analyses were performed to obtain important parameters for freeze-drying process
154
as collapse temperature (Tc), glass transition temperature (Tg) and eutectic temperature (Te) of
155
Ch-5-NFB-NP with the excipients glycine, saccharose and lactose at concentrations of 0, 2.5, 5
156
and 10 %. The Ch-5-NFB-NP was analyzed by DSC - Differential Scanning Calorimetry (Perkin
157
Elmer precisely, DSC 4000). The samples were frozen at -60 ºC and after that, heated to 20 ºC at
158
10 ºC/min to obtain the Tg.
159
To observe the behavior of Ch-5-NFB-NP in freeze-drying process and to obtain the Tc, the
160
samples were analyzed by freeze drying microscopy (FDM) (Lyostat 2, TMS94, Linkam
161
Instruments, Surrey, UK). The samples were frozen to -60 ºC and heated to 10 ºC at 10 ºC/min
162
with 100 mTorr of vacuum.
163
2.7. Nanoparticles freeze-drying
164
Freeze drying was performed in an FTS system TDS-00209-A (SP scientific, Warminster,
165
PA). Ch-5-NFB-NP samples with different concentrations of glycine, lactose and saccharose
166
were freeze-dried at -40 ºC in primary drying and 10 ºC in secondary drying, both at 100 mTorr.
167
The freeze-dried Ch-5-NFB-NP were analyzed by cake appearance; relative moisture using
168
moisture analyzer (Computrac®, Arizona Instrument) and reconstitution time with ultrapure
169
water. DLS and ζ-P measurements were also performed with the samples after reconstitution with
170
ultrapure water to evaluate nanoparticles size, PDI and surface charge.
171
2.8. Scanning Electronic Microscopy
172
One drop of the nanoparticle suspension (Ch-5-NFB-NP and Ch-NP samples) was poured on
173
a glass blade and dried into desiccator in the presence of phosphorus pentoxide. Following, the
174
samples were coated with platinum in equipment Bal-Tec/MED020 in the Laboratory of
175
Technological Characterization (Polytechnique Institute of University of Sao Paulo) and analyzed
176
by Scanning Electronic Microscopy (Quanta 650 FEG, FEI) at 5 kV.
177
2.5. Antimicrobial activity assay
178
S. aureus ATCC 29213, hVISA and ORSA strains were isolated from infected patients at the
179
University Hospital – University of Sao Paulo and maintained frozen in glycerol. Resistance of
180
hVISA and ORSA against oxacillin and vancomycin was determined by disc-diffusion method,
181
using 30 µg of vancomycin and 1 µg of oxacillin.
7
182
The S. aureus strains were cultivated in test tubes with TSB (tripcasein soy beef) for 24 h in
183
a B.O.D. incubator (biochemical oxygen demand) at 35 ºC. After that, 1 mL of each culture was
184
transferred to 4 mL of Mueller-Hington (MH) broth and cultivated in a B.O.D. incubator for 24 h
185
at 35 ºC. The turbidity of the cultures at 580 nm was then adjusted with culture medium to 0.5
186
according to the Mc Farland scale, in which the absorbance must be between 0.08 and 0.1,
187
resulting in a solution of 108 CFU/mL. Samples of 1 mL of this adjusted culture were diluted in
188
99 mL of NaCl 0.9% solution and, subsequently, samples of 1 mL of the diluted solutions were
189
diluted in 99 mL of MH broth, resulting in 104 CFU/mL final inocula. These final inocula were
190
used to minimal inhibitory concentration (MIC) determinations. MIC was determined by
191
colorimetric method using bromocresol purple as dye and mannitol as carbohydrate source [14].
192
Different concentrations of 5-NFB, Ch-5-NFB-NP and Ch-NP were tested against S. aureus
193
ATCC 29213, hVISA and ORSA by microdilution method in 96 wells microplates with MH broth
194
as culture medium, DMSO 4%, bromocresol purple 0.01% and mannitol 0.1%. Microplates were
195
incubated for 18 hours at 37 oC in a B.O.D. incubator and positive and negative controls were
196
present in all tests. The MICs were determined by the color change on the minimal concentration
197
from purple (no bacterial growth) to yellow (bacterial growth). The tests were made in triplicate.
198
The assay was performed with 5-NFB and Ch-5-NFB-NP at drug concentrations from 0.1 to 9
199
µM with 0.5 µM interval. Additionally, Ch-NP tests were performed at the same dilutions used
200
for Ch-5-NFB-NP.
201 202
3. RESULTS AND DISCUSSION
203
3.1. Nanoparticles preparation and characterization
204
The gelation method is based on ionic interactions between the positive charges of chitosan
205
amino groups with negative charges of sodium tripolyphosphate as crosslink agent, forming a
206
polyeletrolytic complex (PEC). Here the gelation method was adapted since the 5-NFB presents a
207
hydrophobic character and this method is more suitable for hydrophilic molecules, due to the
208
hydrophilic solvents used in the process. Therefore, a co-solvent (ethanol) and a surfactant
209
(Polysorbate 20) were used to improve the apparent solubility of 5-NFB, which was determined
210
to be 1 mg/mL. Aiming at biocompatible formulations for future application in burn patients,
211
nontoxic solvents, co-solvents and surfactants were employed.
8
212
According to Wu et al., 2005 [15], NaCl can be used to control size and PDI of chitosan
213
nanoparticles. In this paper, Ch-5-NFB-NP were prepared at different NaCl concentrations and
214
the effect on average size (d, nm), polydispersity index (PDI) encapsulation efficiency (EE%) and
215
ζ-potential can be observed in Table 1.
216 217 218 219 220
Table 1- CH-5-NFB-NP and Ch-NP characterization, as a function of NaCl concentration. Values of average size, polidispersity index, ζ-potential and encapsulation efficiency (EE%) for the chitosan nanoparticles. Error bars correspond to standard deviations of three measurements. System CH-5-NFB-NP CH-NP
NaCl (mM) 0 75 155 0
Average size (d, nm) 322 ± 17 523 ± 51 458 ± 75 320 ± 16
PDI 0.180 ± 0.004 0.230 ± 0.036 0.196 ± 0,04 0.202 ± 0.04
ζ-Potential (mV) EE (%)
+37.3 ± 5.8 +43.4 ± 5.5 +47.1 ± 4.6 +32.3 ± 6.9
44 ± 3 8±2 4±2 -
221 222 223
The target nanoparticle size varies depending on application. For systemic applications,
224
usually nanoparticles of up to 200 nm are desired to avoid nanoparticles accumulation and blood
225
vessels clogging [16]. For topical application such as skin lesions, on the other hand,
226
nanoparticles can present sizes larger than 200 nm to promote drug delivery locally [16].
227
Therefore, the nanoparticles obtained can be considered suitable for skin lesions treatment, since
228
sizes varied from 320 up to 523 nm. All systems presented monomodal size distributions with
229
only one peak for scattering intensity, indicating that systems were reasonably uniform in particle
230
size. Additionally, PDI values were below 0.230, what is considered adequate for pharmaceutical
231
application [17]. Regarding the ζ-Potential, it is accepted that absolute values above 30 mV
232
usually result in colloidal stability in charged systems [17]. The systems developed presented
233
values higher than + 30 mV corresponding to positively charged stable chitosan nanoparticles.
234
The nanoparticle size was found to increase with NaCl addition, probably due to
235
destabilization of nanoparticles charges by electrostatic shielding [18]. With the increased ionic
236
strength, chitosan molecules adopt less extended conformations and the increased shielding effect
237
of counter-ions results in less cross-linking points on chitosan to be accessed by TPP, increasing
238
particles size. The NaCl influence on ζ-potential was less significant and might be explained by
239
the increase in ionic strength at the superficial layer of the nanoparticle that influences chitosan
240
electrophoretic mobility [19].
9
241
As can also be seen in Table 1, encapsulation efficiency decreased with NaCl concentration,
242
what was not expected since nanostructures presented increased size with increasing salt
243
concentrations. We believe the salt might have an effect on polysorbate micelles that in turn are
244
responsible for 5-NFB solubilization. Since Polysorbate 20 concentration in the systems is above
245
its critical micellar concentration (CMC = 0.06 mM, Daltin, 2011), micelles are supposed to be
246
present. Owing to the hydrophobic effect, one would expect 5-NFB to partition preferentially to
247
the hydrophobic compartment of polysorbate micelles. In fact, polysorbate micelles were
248
identified in the system by DLS before TPP addition, with average diameters of 13.3 nm (empty
249
micelles) and 12.2 nm (micelles in the presence of 5-NFB). Therefore, we assumed that 5-NFB is
250
solubilized in Polysorbate micelles, which are further incorporated in chitosan nanoparticles. To
251
better prove our hypothesis, we compared chitosan nanoparticles prepared in the absence of
252
Polysorbate 20 with the Ch-NP with and without the 5-NFB (Table 2). As can be seen, an
253
increase in the nanoparticles size is observed in the presence of Polysorbate 20, probably due to
254
the chitosan gelation process in the presence of the surfactant micelles that are incorporated.
255
Regarding the NaCl effect on the micelles, it is well known that nonionic micelles of surfactants
256
with poly(ethylene oxide) (PEO) head groups suffer influence of salt addition due to the
257
dehydration of the PEO corona, since water molecules would rather interact with NaCl ions [20].
258
This effect might decrease the internal micellar volume for 5-NFB solubilization. Additionally,
259
increasing NaCl concentration might also lower the compound apparent water solubility.
260 261 262 263
Table 2- Chitosan nanoparticles (CH-NP) average size and polydispersity index (PDI) in the presence or not of polysorbate 20 (P20). Error bars correspond to standard deviations of three measurements. System CH-NP without P20 CH-NP with P20 CH-5-NFB-NP with P20
Average size (d, nm) 209.2 ± 1.7 320.2 ± 3.1 321.5 ± 3.2
PDI 0.157 ± 0.02 0.202 ± 0.01 0.180 ± 0.01
264 265
Our results show the influence of salt concentration in chitosan nanoparticles size, PDI
266
and zeta potential. More important, we show that for systems with nonionic surfactants, salt
267
addition is deleterious, especially if the drug is preferentially incorporate in the surfactant
268
micelles. Also, we show that chitosan nanoparticles can be an alternative for hydrophobic drugs
269
incorporation when the drug is initially solubilized in surfactant micelles.
10
270
3.2. Thermal analyses
271
The DSC analyses were performed with Ch-5-NFB-NP at different concentrations of
272
saccharose, lactose and glycine (0, 2.5, 5 and 10%) to determine the glass transition temperature
273
(Tg’) and eutectic temperature (Teut) (Table 3). The lyophilization with optical microscopy was
274
performed with the samples to determine the collapse temperature (Tcol). The Tg’ is defined by
275
the transition of viscous state (bloody) to stiff state of amorphous solid in freezing process, with
276
viscosity increase. The Teut is the collapse temperature of crystalline compounds between
277
freezing and melting point of these compounds. The Tcol is the temperature that the frozen
278
structure does not support itself in the primary drying because of high water mobility [21].
279
Determination of Tg’, Teut and Tcol is very important for freeze-drying processes because
280
the amorphous or crystalline solid have to be in stiff state during primary freeze-drying. If the
281
primary drying occurs above Tg’, Teut and Tcol, the water is sufficient free in interstitial region
282
and can no longer keep its original configuration, leading to a collapse duo to low matrix
283
viscosity above these temperatures. So, the freeze-drying has to occur below Tg’, Teut and Tcol.
284
According to DSC analyses, the sample CH-5-NFB-NP 2.5% glycine did not present a Tg’. The
285
samples CH-NP, Ch-5-NFB-NP 10% lactose, CH-5-NFB-NP 10% glycine and all concentrations
286
of CH-5-NFB-NP saccharose did not present Teut. The Teut demonstrated in CH-5-NFB-NP
287
evidence the crystalline character of 5-NFB in CH-5-NFB-NP.
288 289
The Tcol was determined base optical cryomicroscopy images (Figure 2) to preview the minimal temperature that freeze-drying process have to occur without “cake” collapse.
11
290 291 292 293 294 295 296 297 298 299
Figure 2- Microscope images of lyophilization process of CH-5-NFB-NP without excipients. a) liquid structure; b) frozen structure; c) freeze-drying layer; d) microcollapse; e) macrocollapse; f) total collapse. This experiment was performed with glycine, lactose and saccharose at 2.5, 5 and 10%.
Table 3- Values of Teut, Tg’and Tcol (°C) for CH-5-NFB-NP with lactose, saccharose, glycine and water as excipients. Excipient
Tcol
-3.4 -2.4 -
-38.8 -39.9 -44.4 -35.0
-35.5 -28.0 -37.3 -26.4
5% 2.5% 10%
-
-35.0 -37.0 -37.0
-26.6 -23.2 -3.9
5% 2.5%
-5.2 -7.8
-24.0 -
-11.3 -38.5
-
1.4
-
-24.3
Saccharose
Glycine
Lactose
Water
300
Tg’
Concentration 10% 5% 2.5% 10%
Teut
12
301
In freeze-drying process, the higher the Tg’ and Tcol’ temperatures the faster and cheaper is
302
the process. Temperatures below -40 °C are not viable, since the process becomes slow and
303
expansive. The higher Tg’ was observed with Ch-5-NFB-NP 5% glycine (Tg’= -24 °C) and the
304
higher Tcol was observed with CH-5-NFB-NP 10% glycine (-3.9 °C).
305
3.3 Freeze-drying
306
In freeze-drying process excipients are usually employed as bulking agents, lyoprotectants
307
and cryoprotectants. Among them, one can cite polysaccharides, saccharides, proteins and
308
aminoacids. The bulking agents are responsible to provide volume to dry matrix, replacing the
309
water and forming a crystalline structure that supports the cake. Additionally, cryoprotectants
310
protect the active agent during freeze-drying process [20]. The primary freeze-drying process
311
occurred with about 20 hours and the cakes were kept under vacuum (Figure 3).
312
1
2
3
4 313 314 315 316 317
Figure 3- Lyophilized cakes of CH-5-NFB-NP with different excipients. Line 1: water; Line 2: glycine at 2.5, 5 and 10%; Line 3: lactose at 2.5, 5 and 10%; Line 4: 2.5, 5 and 10%.
13
318
The glycine cakes were adequate, with no shrinkages and cracks, considered ideal to the
319
formulation preservation, transportation and commercialization. The CH-5-NFB-NP lyophilized
320
cakes with lactose and saccharose were acceptable, but presented shrinkages and cracks, most of
321
them not associated with collapse. These phenomena are related with stress in primary drying, in
322
which the water in the sample leaves the structure causing stress and forming shrinkages and/or
323
cracks. Shrinkages are usually associated to low concentrations of bulking agents and cracks are
324
associated to high concentrations. However, these events are acceptable and do not interfere in
325
product quality, except to possible cake deconfiguration during transportation [22].
326
All cakes were immediately resuspended in ultrapure water and the characteristics of the
327
reconstituted systems are presented in Table 4. Glycine is widely employed as bulking agent in
328
freeze-drying process due to its low Tg’ and Tcol’, resulting in elegant cakes. Additionally,
329
glycine is safe and biocompatible as an excipient. In our case glycine did not result in adequate
330
systems after the nanoparticles reconstitution. The CH-5-NFB-NP with glycine 2.5% and 5%
331
presented aggregates a few minutes after reconstitution, as can be seen for the high PDI values
332
observed for both samples. Only the highest concentration of glycine (10%) was able to stabilize
333
the CH-5-NFB-NP, visually preserving colloidal stability. In neutral pH, glycine is totally ionized
334
and the negative charges of the carboxylic groups can electrostatically interact with the positive
335
nanoparticles surface. As a consequence, glycine may shields the nanoparticles surface resulting
336
in lower zeta potential value, since the amine groups of glycine that will be at the nanoparticles
337
surface are weaker than amine groups of chitosan. At lower zeta potential values, nanoparticles
338
aggregation was observed.
339 340
Table 4- Lyophilized CH-5-NFB-NP characteristics after resuspension. Lyophilized Sample
Average size (d.nm)
PDI
Zeta Potential (mV)
CH-5-NFB-NP
Not resuspend
-
-
Relative Moisture (%) 3.5
CH-5-NFB-NP
607.0 ± 21.5
glycine 2.5%
(aggregates)
0.765 ± 0.19
+12.9 ± 4.75
3.55
CH-5-NFB-NP
352.4 ± 5.23
glycine 5%
(aggregates)
0.440 ± 0.09
+16.4 ± 4.64
2.49
14
CH-5-NFB-NP glycine 10% CH-5-NFB-NP lactose 2.5% CH-5-NFB-NP lactose 5% CH-5-NFB-NP lactose 10% CH-5-NFB-NP saccharose 2.5% CH-5-NFB-NP saccharose 5% CH-5-NFB-NP saccharose 10%
305.1 ± 3.24
0.251 ± 0.021
+20.3 ± 4.31
2.18
295.5 ± 4.21
0.239 ± 0.034
+31.1 ± 5.31
3.67
299.2 ± 2.49
0.228 ± 0.018
+29.7 ± 4.24
2.80
336.7 ± 3.29
0.259 ± 0.016
+20.9 ± 4.68
1.38
317.4 ± 2.23
0.233 ± 0.019
+33.0 ± 6.26
5.41
331.1 ± 4.5
0.229 ± 0.035
+31.8 ± 5.13
3.73
347.1 ± 2.56
0.253 ± 0.024
+22.6 ± 4.39
3.13
341 342
Both saccharose and lactose were able to preserve the nanoparticles characteristics at all
343
concentrations, except the saccharose 2.5% that resulted in a high moisture (5.41 %) for
344
lyophilized products[20]. For these lyoprotectants, the only interactions are the hydrogen bonds
345
among water molecules and the nanoparticles. Based on our results and previous ones by Allison,
346
Brynildsen and Collins et al. (2011)[23], we believe that saccharose at 5 and 10% can be
347
considered the best lyoprotectant since it demonstrated to be useful for bacterial resistance
348
combat, in which the saccharides were responsible to “wake-up” the latent bacteria, facilitating
349
the bactericidal agents activity.
350
3.4 Scanning Electron Microscopy, (SEM)
351
The morphology of CH-NP and Ch-5-NFB-NP were observed by SEM and, based on the
352
images obtained (Figure 4), both CH-NP and Ch-5-NFB-NP can be considered spherical and with
353
an average diameter of 450 nm, what is significantly larger that the values obtained by DLS
354
measurements (320 nm). We believe this difference might result from the staining process and
355
also from some degree of polymer melting during SEM analysis, owing to the increase in sample
15
356
temperature with the laser beam incidence on the nanoparticles. Nonetheless, SEM is important
357
to confirm the nanoparticles shape, whereas DLS is more adequate to estimate the polymer
358
nanoparticles average size.
359 360 361
Figure 4- Scanning electron microscopy images. a) CH-NP (scale bar = 10 µm) and b) CH-5NFB-NP (scale bar = 5 µm).
362 363
3.5. Antimicrobial activity assay
364
The antimicrobial activity of 5-NFB, CH-5-NFB-NP and CH-NP 5-NFB was determined
365
against strains of S. aureus ATCC 29213, hVISA and ORSA by minimal inhibitory concentration
366
(MIC) and results are presented in Table 5.
367 368 369 370 371
372 373
Table 5- Minimal inhibitory concentration of 5-NFB, CH-5-NFB-NP and CH-NP against S. aureus strains ATCC 29213, hVISA and ORSA, considering 5-NFB MIC present into nanoparticles and nanoparticles MIC (Nps). Error bars correspond to standard deviations of three measurements. Minimal Inhibitory Concentration (MIC)
1.4 – 1.5
CH-5-NFB-NP 5-NFB MIC (µg/mL) 0.4 – 0.5
CH-5-NFB-NP Nps MIC (µg/mL) 10.9 – 14.6
CH-NP Nps MIC (µg/mL) 24.3 – 40.4
Hvisa
1.4 – 1.5
0.8 – 0.9
14.6 – 21.9
24.3 – 32.3
ORSA
2.2 – 2.3
0.6 – 0.8
18.3 – 21.9
24.3 – 32.3
S. aureus strain
5-NFB (µg/mL)
ATCC 29213
16
374
According to the results, 5-NFB showed higher activity against ATCC 29213 and hVISA
375
strains and was less active against ORSA. Previously, our group demonstrated that the 5-NFB
376
activity against ATCC 29213 (5.8 µM) is superior to nifuroxazide (16.0 – 4.0 µM) and
377
vancomycin (<20 µM) [3]. The CH-5-NFB-NP activity was 3 times higher than 5-NFB (in
378
relation to CH-5-NFB-NP/ 5-NFB MIC) and 2 times higher than CH-NP (in relation to CH-5-
379
NFB-NP/ Nps MIC) against ATCC 29213; 2 times higher than 5-NFB (in relation to CH-5-NFB-
380
NP/ 5-NFB MIC) and 1.7 times higher than CH-NP (in relation to Ch-5-NFB-NP/ Nps MIC)
381
against hVISA; 3 times higher than 5-NFB (in relation to CH-5-NFB-NP/ 5-NFB MIC) and 1.3
382
times higher than CH-NP against ORSA (in relation to CH-5-NFB-NP/ Nps MIC). Our results
383
point to a synergistic effect of 5-NFB and chitosan nanoparticles since the concentration of
384
compound and chitosan nanoparticles in the CH-5-NFB-NP necessary to inhibit the bacterial
385
growth is lower than 5-NFB and Ch-NP isolated effects. The antibacterial mechanism of 5-NFB
386
is related to reduction of nitro group in toxic radicals that interferes in DNA and RNA cellular
387
synthesis [3]; the antibacterial mechanism of chitosan, on the other hand, is related to its
388
protonated amino groups that may interact with negative charges of bacterial proteins and other
389
cellular constituents [8]. This is very interesting regarding antibacterial resistance, when two or
390
more antibacterial agents used together with distinct mechanisms may decrease the antimicrobial
391
resistance [24]. These results demonstrated that this formulation could be effective to eliminate
392
resistant infections.
393 394
4. CONCLUSIONS
395
In this paper, we present a novel formulation for a promising antibacterial compound, 5-
396
NFB: N’-((5-nitrofuran-2-yl)methylene)-2-benzhydrazide. Since it is a hydrophobic compound,
397
we solubilized it in Polysorbate 20 micelles that were further incorporated in chitosan
398
nanoparticles. The CH5-NFB-NP presented a diameter of 321 nm, PDI of 0.18, ζP +37 mV and
399
an encapsulation efficiency of 44%, resulting in good parameters to topical application and
400
nanoparticles stability. Samples were successfully lyophilized using saccharose or lactose as bulk
401
agents. and SEM analyses showed spherical and regular nanoparticles. The CH-5-NFB-NP
402
presented increased activity in comparison to the free compound and empty chitosan
403
nanoparticles (CH-NP) against all S. aureus strains, including multidrug-resistant strains (hVISA
404
and ORSA). In addition, our results point to a synergic antimicrobial activity between chitosan
17
405
nanoparticles and 5-NFB. Therefore, CH-5-NFB-NP demonstrated to be a promising alternative
406
for the treatment of S. aureus infections, especially in skin burned areas. On a broader
407
perspective, several papers present interesting results in the design and preparation of chitosan
408
nanoparticles. However, they are usually designed for the incorporation of drugs based on
409
entrapment and/or electrostatic interactions of the drugs with the cross-linked chitosan net. This
410
is the first time a complex system based on the encapsulation of a hydrophobic drug into
411
Polysorbate micelles with further entrapment in chitosan nanoparticles is described. It opens the
412
possibility of other hydrophobic drugs incorporation into similar systems.
413
Acknowledgments
414
The authors acknowledge the financial support of the Coordination for the Improvement of
415
Higher Education Personnel - CAPES (Process 001) and the National Council for Scientific
416
and Technological Development-CNPQ.
417
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Highlights ““Chitosan nanoparticles for the delivery of a new compound active against multidrugresistant Staphylococcus aureus” Laura Freitas de Andrade, Alexsandra Conceição Apolinário, Carlota de Oliveira RangelYagui, Marco Antonio Stephano, Leoberto Costa Tavares. • • •
The efficient loading of lipophilic compound 5-NFB into chitosan nanoparticles; Efficient lyophilization of Ch-5-NFB-NP with lactose and sacharose as bulk agents; Great activity of Ch-5-NFB-NP against multidrug resistant Staphylococcus aureus.
Conflict of Interest Statement
“Chitosan nanoparticles for the delivery of a new compound active against multidrugresistant Staphylococcus aureus” The authors whose names are listed immediately below certify that they have NO affiliations with or involvement in any organization or entity with any financial interest (such as honoraria; educational grants; participation in speakers’ bureaus; membership, employment, consultancies, stock ownership, or other equity interest; and expert testimony or patent-licensing arrangements), or non-financial interest (such as personal or professional relationships, affiliations, knowledge or beliefs) in the subject matter or materials discussed in this manuscript. 1. 2. 3. 4. 5.
Laura Freitas de Andrade Alexsandra Conceição Apolinário Carlota de Oliveira Rangel-Yagui Marco Antonio Stephano Leoberto Costa Tavares
S1773-2247(19)31087-1
DOI:
https://doi.org/10.1016/j.jddst.2019.101363
Reference:
JDDST 101363
To appear in:
Journal of Drug Delivery Science and Technology
Received Date: 31 July 2019 Revised Date:
21 October 2019
Accepted Date: 28 October 2019
Please cite this article as: L.F. de Andrade, Alexsandra.Conceiçã. Apolinário, C. de Oliveira RangelYagui, Marco.Antô. Stephano, L.C. Tavares, Chitosan nanoparticles for the delivery of a new compound active against multidrug-resistant Staphylococcus aureus, Journal of Drug Delivery Science and Technology (2019), doi: https://doi.org/10.1016/j.jddst.2019.101363. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2019 Published by Elsevier B.V.
1 2
Chitosan nanoparticles for the delivery of a new compound active against multidrug-resistant Staphylococcus aureus
3 4 5 6
Laura Freitas de Andrade, Alexsandra Conceição Apolinário, Carlota de Oliveira Rangel-Yagui, Marco Antônio Stephano, Leoberto Costa Tavares*
7 8
Department of Biochemical and Pharmaceutical Technology, Faculty of Pharmaceutical Sciences, University of Sao Paulo, São Paulo, Brazil.
9
**Correspondence Author:
10
[email protected]
11
University of Sao Paulo
12
Av. Prof. Lineu Prestes, 580
13
05508-000 - São Paulo, SP, Brazil
14
+ 55 11 30912385
15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33
Abstract: Chitosan is a biopolymer with antimicrobial, analgesic activity, tissue regenerator properties and biofilm protection. The N’-((5-nitrofuran-2-yl)methylen)-2-benzhydrazide, a novel active compound against multidrug-resistant Staphylococcus aureus (5-NFB), was incorporated in Polysorbate 20 micelles and further loaded in chitosan nanoparticles (Ch-5-NFB-NP), prepared by ionic gelation varying NaCl concentration. The nanoparticles were characterized by Dynamic Light Scattering (DLS) to determine size, polydispersity index (PDI) and ζ-potential. Encapsulation Efficiency (EE%) was determined by indirect method and morphology by scanning electronic microscopy (SEM). Antimicrobial activity tests against Staphylococcus aureus strains ATCC 29213, hVISA and ORSA were performed with 5-NFB, Ch-5-NFB-NP and empty chitosan nanoparticles (Ch-NP) using colorimetric and microdilution methods by minimal inhibitory concentration (MIC). In the optimal experiment, the Ch-5-NFB-NP were obtained with average diameter of 321 nm, PDI of 0.18, ζ-P of +37 mV; EE% of 44% and the morphology by SEM showed spherical and regular shaped nanoparticles. The best results for bacterial growth inhibition against all strains tested were observed for Ch-5-NFB-NP. The nanoparticles were lyophilized with different lyoprotectants and the best freeze-dried samples were obtained with lactose and saccharose, keeping the Ch-5-NFB-NP characteristics. Therefore, owing to the antibacterial activity, tissue regenerator property and protective biofilm effect, the Ch-5-NFB-NP are a promising alternative to treat multidrug-resistant infections, especially in burned patients.
34 35 36
Keywords: Nifuroxazide; Chitosan; Nanoparticles; Drug delivery; Multidrug-resistant bacteria; Lyophilization.
2
37
1. INTRODUCTION
38
Antibiotic resistance is one of the main concerns in global health and bacterial resistant
39
strains results in many deaths in hospitals [1]. Infections by multidrug-resistant bacteria is
40
responsible for 700 thousand deaths per year worldwide and this number may increase giving the
41
indiscriminate use of antibiotics in therapeutics and farming, as well as incorrect discharge of
42
these substances[1]. Among the most threatening bacteria, one can highlight penicillin resistant
43
Staphylococcus epidermidis and Staphylococcus aureus, in particular the following strains:
44
methicilin resistant S. aureus (MRSA), oxacillin resistant S. aureus (ORSA), vancomycin
45
intermediate S. aureus (VISA) and heterogeneous vancomycin intermediate S. aureus (hVISA).
46
Multidrug-resistant strains of anaerobic and facultative aerobic bacteria as S. aureus usually
47
infect burns and cutaneous lesions of large extension, leading to high rates of sepsis in patients
48
and, consequently, high morbidity and mortality [2]. Within this context, novel molecules and/or
49
pharmaceutical formulations with significative activity against these resistant strains are of great
50
interest.
51
Our group has recently synthesized a 5-nitro-2-heterocyclic compound analog to
52
nifuroxazide
(Fig.
1)
that
presented
significant
activity
against
multidrug-resistant
53
microorganisms as S. aureus, S. epidermidis and C. albicans [3]. Although the mechanism of
54
action of this class is not fully elucidated, the bactericidal effect seems to be related to the
55
reduction of the nitro group (-NO2) leading to the generation of free radicals that, in turn,
56
interfere with the cellular metabolism, as DNA and RNA structures and mitochondrial
57
respiration. The nitro-group is more selective to bacterial, fungal and protozoan cells than human
58
cells and the major reduction potential of nitro group is directly linked to increased cellular death
59
[4].
60
61 62 63
Figure 1- Chemical structure of nifuroxazide and its 5-NFB analog: N’-((5-nitrofuran-2-
64
yl)methylene)-2-benzhydrazide.
65
3
66
The nifuroxazide analog N’-((5-nitrofuran-2-yl)methylene)-2-benzhydrazide (5-NFB) can be
67
considered a promising alternative to treat infections by multidrug-resistant microorganisms, it
68
was one of the most potent among the nitro-compound series previously studied [3]. However,
69
the high lipophilicity hinders further evaluation and potential application of this molecule. In this
70
sense, incorporation into nanostructures can be an alternative for the delivery of 5-NFB by
71
promoting higher solubility and improving bioavailability. Additionally, topical administration of
72
nanostructured antimicrobials is advantageous for effective treatment of local disease, such as
73
burned areas, due to their inherent ability to circumvent systemic cytotoxicity and ease of rapid
74
delivery at the site of infection [5,6].
75
Nanostructures based on chitosan biopolymer have been widely studied as drug delivery
76
systems owing to its biocompatibility and low toxicity [7]. Chitosan is an oligomer formed by D-
77
glucosamine and D-glucoacetamide units linked by glucosides β-(1 > 4) bonds (Fig. 2). It is
78
obtained by chitin deacetylation, which provides the polycationic character of chitosan by amines
79
protonation in acid environment [8]. This polymer is able to bind polyanions as sodium
80
tripolyphosphate (TPP), forming hydrogels that can be employed as biofilms, nanoparticles and
81
microparticles [8,9].
82
One interesting point of using chitosan as a biomaterial for nanostructures preparation is that
83
it has analgesic, healing and antibacterial pharmacological activities. The antimicrobial activity of
84
nicin and natamycin incorporated into chitosan nanoparticles, for example, was proved against
85
Salmonella enterica and Penicillium chrysogenum, with 50% and 12.5% of reduction in MIC
86
compared to free-nicin and free-natamycin [11]. Additionally, it presents mucoadhesive
87
proprieties with biofilm formation. All these properties have been encouraging the investigation
88
of chitosan as scaffold for tissue regeneration. The antibacterial activity of chitosan films and
89
chitosan nanoparticles is related to the positive charges of chitosan that bind to the negative
90
charges in bacteria, but the mechanisms of action is not fully known [10]. Chitosan nanoparticles
91
have also been widely studied to increase apparent solubility and bioavailability of drugs. The
92
mucoadhesive property and biofilm formation of chitosan enable higher and prolonged contact to
93
the administration route [12,13], what is of great interest for skin infections such as the ones
94
caused by S. aureus strains.
95
In this paper, we developed chitosan nanoparticles loaded with N’-((5-nitrofuran-2-
96
yl)methylene)-2-benzhydrazide (Ch-5-NFB-NP) by ionic gelation method aiming an stable
4
97
delivery systems. The 5-NFB-loaded chitosan NP were proved to be active in vitro against S.
98
aureus ATCC 29213, hVISA and ORSA strains. Additionally, we investigated the lyophilization
99
process for the nanoparticles developed using glycine, lactose and saccharose as lyoprotectants.
100 101
2. MATERIALS AND METHODS
102
2.1. Materials
103
Chitosan (low molecular weight of 50 – 190 kDa and deacetylaytion degree of 75 – 85%),
104
sodium tripolyphosphate (85% of purity), Mueller Hington broth and bromocresol purple were
105
purchased from Sigma Aldrich (St. Louis, MO, USA). N’-((5-nitrofuran-2-yl)methylen)-2-
106
benzhydrazyde compound (5-NFB) was previously synthesized by our group and duly purified by
107
DMF recrystallization and identified by NMR H1 and NMR C13 (Zorzi et al., 2014). All other
108
reagents were purchased from LabSynth (Diadema, SP, BRA).
109
2.2. Preparation of empty chitosan nanoparticles (Ch-NP) and chitosan/5-NFB nanoparticles
110
(Ch-5-NFB-NP)
111
The Ch-NP and Ch-5-NFB-NP preparation was adapted from a previously described method
112
[11]. Briefly, chitosan (2 mg/mL) was dissolved in acetic acid 1% (pH 5.2); ethanol (10%) and
113
Polysorbate 20 (1.4 mM) were added in the solution. Ch-NP were produced by the dropwise
114
addition of a TPP solution (1 mg/mL) up to 3:1 chitosan/TPP ratio, under magnetic stirring at
115
room temperature during 1 h. For the Ch-5-NFB-NP, a solution of 5-NFB in ethanol (1 mg/mL)
116
was slowly added to the chitosan/ethanol/Polysorbate 20 solution under magnetic stirring before
117
TPP addition. The effect of ionic strength on the Ch-5-NFB-NP colloidal stability was
118
investigated by varying the NaCl concentration (0.75 and 155 mM) in the chitosan and TPP
119
solutions.
120
NPs were purified by centrifugation at 13,000 x g in 10 µL of pure glycerol as support into
121
Eppendorf flasks for 15 minutes. The supernatant was used for indirect determination of the drug
122
encapsulation efficiency (EE%) and the precipitated nanoparticles were resuspended with
123
ultrapure water completing the previous volume.
124
2.3. Nanoparticles Characterization
5
125
The NPs samples were prepared as explained above and they were analyzed by photon
126
correlation spectroscopy (Dynamic Light Scattering) to determine average size and zeta potential
127
both using the Zetasizer Nano ZS (Malvern Instruments Ltd, Worcestershire,UK). The size and
128
polydispersity index were measured in an angle of 175º and 532 nm laser, using glass cuvettes.
129
Stokes Einstein equation provided the hydrodynamic diameter from diffusion coefficient values
130
(Equation 1). In addition, particle size distribution was provided by cumulant analysis method.
131
The mobility was then related to the ζ-potential using the Henry equation (Equation 2). All
132
measurements were performed at least three times.
133 =
134
(Equation 1)
135
in which: kB = Boltzmann constant (1.38064852 × 10 − 23 J/K), T = temperature, η = absolute
136
viscosity of the PBS and Rh = hydrodynamic radius.
137 UE =
138
(
)
(Equation 2)
139
in which: UE = electrophoretic mobility, z = zeta potential, ε = dielectric constant, η = viscosity
140
and f(κa) = Henry’s function.
141 142
2.4. Encapsulation efficiency
143
The percentage of encapsulation efficiency (EE%) was calculated indirectly from the
144
difference between the initial total amount of 5-NFB added to the system and the amount of 5-
145
NFB in the supernatant after centrifugation (free compound), as presented in Eq. 3. The 5-NFB
146
concentration in supernatant was measured by UV spectrophotometry at 370 nm, in a UV/VIS
147
spectrophotometry (Spectra Max Plus 384– Molecular Devices).
148 149
EE% = Total amount of 5-NFB – Supernatant amount of 5-NFB x 100
150 151 152
Total amount of 5-NFB 2.6. Thermal analyses
(Equation 3)
6
153
Thermal analyses were performed to obtain important parameters for freeze-drying process
154
as collapse temperature (Tc), glass transition temperature (Tg) and eutectic temperature (Te) of
155
Ch-5-NFB-NP with the excipients glycine, saccharose and lactose at concentrations of 0, 2.5, 5
156
and 10 %. The Ch-5-NFB-NP was analyzed by DSC - Differential Scanning Calorimetry (Perkin
157
Elmer precisely, DSC 4000). The samples were frozen at -60 ºC and after that, heated to 20 ºC at
158
10 ºC/min to obtain the Tg.
159
To observe the behavior of Ch-5-NFB-NP in freeze-drying process and to obtain the Tc, the
160
samples were analyzed by freeze drying microscopy (FDM) (Lyostat 2, TMS94, Linkam
161
Instruments, Surrey, UK). The samples were frozen to -60 ºC and heated to 10 ºC at 10 ºC/min
162
with 100 mTorr of vacuum.
163
2.7. Nanoparticles freeze-drying
164
Freeze drying was performed in an FTS system TDS-00209-A (SP scientific, Warminster,
165
PA). Ch-5-NFB-NP samples with different concentrations of glycine, lactose and saccharose
166
were freeze-dried at -40 ºC in primary drying and 10 ºC in secondary drying, both at 100 mTorr.
167
The freeze-dried Ch-5-NFB-NP were analyzed by cake appearance; relative moisture using
168
moisture analyzer (Computrac®, Arizona Instrument) and reconstitution time with ultrapure
169
water. DLS and ζ-P measurements were also performed with the samples after reconstitution with
170
ultrapure water to evaluate nanoparticles size, PDI and surface charge.
171
2.8. Scanning Electronic Microscopy
172
One drop of the nanoparticle suspension (Ch-5-NFB-NP and Ch-NP samples) was poured on
173
a glass blade and dried into desiccator in the presence of phosphorus pentoxide. Following, the
174
samples were coated with platinum in equipment Bal-Tec/MED020 in the Laboratory of
175
Technological Characterization (Polytechnique Institute of University of Sao Paulo) and analyzed
176
by Scanning Electronic Microscopy (Quanta 650 FEG, FEI) at 5 kV.
177
2.5. Antimicrobial activity assay
178
S. aureus ATCC 29213, hVISA and ORSA strains were isolated from infected patients at the
179
University Hospital – University of Sao Paulo and maintained frozen in glycerol. Resistance of
180
hVISA and ORSA against oxacillin and vancomycin was determined by disc-diffusion method,
181
using 30 µg of vancomycin and 1 µg of oxacillin.
7
182
The S. aureus strains were cultivated in test tubes with TSB (tripcasein soy beef) for 24 h in
183
a B.O.D. incubator (biochemical oxygen demand) at 35 ºC. After that, 1 mL of each culture was
184
transferred to 4 mL of Mueller-Hington (MH) broth and cultivated in a B.O.D. incubator for 24 h
185
at 35 ºC. The turbidity of the cultures at 580 nm was then adjusted with culture medium to 0.5
186
according to the Mc Farland scale, in which the absorbance must be between 0.08 and 0.1,
187
resulting in a solution of 108 CFU/mL. Samples of 1 mL of this adjusted culture were diluted in
188
99 mL of NaCl 0.9% solution and, subsequently, samples of 1 mL of the diluted solutions were
189
diluted in 99 mL of MH broth, resulting in 104 CFU/mL final inocula. These final inocula were
190
used to minimal inhibitory concentration (MIC) determinations. MIC was determined by
191
colorimetric method using bromocresol purple as dye and mannitol as carbohydrate source [14].
192
Different concentrations of 5-NFB, Ch-5-NFB-NP and Ch-NP were tested against S. aureus
193
ATCC 29213, hVISA and ORSA by microdilution method in 96 wells microplates with MH broth
194
as culture medium, DMSO 4%, bromocresol purple 0.01% and mannitol 0.1%. Microplates were
195
incubated for 18 hours at 37 oC in a B.O.D. incubator and positive and negative controls were
196
present in all tests. The MICs were determined by the color change on the minimal concentration
197
from purple (no bacterial growth) to yellow (bacterial growth). The tests were made in triplicate.
198
The assay was performed with 5-NFB and Ch-5-NFB-NP at drug concentrations from 0.1 to 9
199
µM with 0.5 µM interval. Additionally, Ch-NP tests were performed at the same dilutions used
200
for Ch-5-NFB-NP.
201 202
3. RESULTS AND DISCUSSION
203
3.1. Nanoparticles preparation and characterization
204
The gelation method is based on ionic interactions between the positive charges of chitosan
205
amino groups with negative charges of sodium tripolyphosphate as crosslink agent, forming a
206
polyeletrolytic complex (PEC). Here the gelation method was adapted since the 5-NFB presents a
207
hydrophobic character and this method is more suitable for hydrophilic molecules, due to the
208
hydrophilic solvents used in the process. Therefore, a co-solvent (ethanol) and a surfactant
209
(Polysorbate 20) were used to improve the apparent solubility of 5-NFB, which was determined
210
to be 1 mg/mL. Aiming at biocompatible formulations for future application in burn patients,
211
nontoxic solvents, co-solvents and surfactants were employed.
8
212
According to Wu et al., 2005 [15], NaCl can be used to control size and PDI of chitosan
213
nanoparticles. In this paper, Ch-5-NFB-NP were prepared at different NaCl concentrations and
214
the effect on average size (d, nm), polydispersity index (PDI) encapsulation efficiency (EE%) and
215
ζ-potential can be observed in Table 1.
216 217 218 219 220
Table 1- CH-5-NFB-NP and Ch-NP characterization, as a function of NaCl concentration. Values of average size, polidispersity index, ζ-potential and encapsulation efficiency (EE%) for the chitosan nanoparticles. Error bars correspond to standard deviations of three measurements. System CH-5-NFB-NP CH-NP
NaCl (mM) 0 75 155 0
Average size (d, nm) 322 ± 17 523 ± 51 458 ± 75 320 ± 16
PDI 0.180 ± 0.004 0.230 ± 0.036 0.196 ± 0,04 0.202 ± 0.04
ζ-Potential (mV) EE (%)
+37.3 ± 5.8 +43.4 ± 5.5 +47.1 ± 4.6 +32.3 ± 6.9
44 ± 3 8±2 4±2 -
221 222 223
The target nanoparticle size varies depending on application. For systemic applications,
224
usually nanoparticles of up to 200 nm are desired to avoid nanoparticles accumulation and blood
225
vessels clogging [16]. For topical application such as skin lesions, on the other hand,
226
nanoparticles can present sizes larger than 200 nm to promote drug delivery locally [16].
227
Therefore, the nanoparticles obtained can be considered suitable for skin lesions treatment, since
228
sizes varied from 320 up to 523 nm. All systems presented monomodal size distributions with
229
only one peak for scattering intensity, indicating that systems were reasonably uniform in particle
230
size. Additionally, PDI values were below 0.230, what is considered adequate for pharmaceutical
231
application [17]. Regarding the ζ-Potential, it is accepted that absolute values above 30 mV
232
usually result in colloidal stability in charged systems [17]. The systems developed presented
233
values higher than + 30 mV corresponding to positively charged stable chitosan nanoparticles.
234
The nanoparticle size was found to increase with NaCl addition, probably due to
235
destabilization of nanoparticles charges by electrostatic shielding [18]. With the increased ionic
236
strength, chitosan molecules adopt less extended conformations and the increased shielding effect
237
of counter-ions results in less cross-linking points on chitosan to be accessed by TPP, increasing
238
particles size. The NaCl influence on ζ-potential was less significant and might be explained by
239
the increase in ionic strength at the superficial layer of the nanoparticle that influences chitosan
240
electrophoretic mobility [19].
9
241
As can also be seen in Table 1, encapsulation efficiency decreased with NaCl concentration,
242
what was not expected since nanostructures presented increased size with increasing salt
243
concentrations. We believe the salt might have an effect on polysorbate micelles that in turn are
244
responsible for 5-NFB solubilization. Since Polysorbate 20 concentration in the systems is above
245
its critical micellar concentration (CMC = 0.06 mM, Daltin, 2011), micelles are supposed to be
246
present. Owing to the hydrophobic effect, one would expect 5-NFB to partition preferentially to
247
the hydrophobic compartment of polysorbate micelles. In fact, polysorbate micelles were
248
identified in the system by DLS before TPP addition, with average diameters of 13.3 nm (empty
249
micelles) and 12.2 nm (micelles in the presence of 5-NFB). Therefore, we assumed that 5-NFB is
250
solubilized in Polysorbate micelles, which are further incorporated in chitosan nanoparticles. To
251
better prove our hypothesis, we compared chitosan nanoparticles prepared in the absence of
252
Polysorbate 20 with the Ch-NP with and without the 5-NFB (Table 2). As can be seen, an
253
increase in the nanoparticles size is observed in the presence of Polysorbate 20, probably due to
254
the chitosan gelation process in the presence of the surfactant micelles that are incorporated.
255
Regarding the NaCl effect on the micelles, it is well known that nonionic micelles of surfactants
256
with poly(ethylene oxide) (PEO) head groups suffer influence of salt addition due to the
257
dehydration of the PEO corona, since water molecules would rather interact with NaCl ions [20].
258
This effect might decrease the internal micellar volume for 5-NFB solubilization. Additionally,
259
increasing NaCl concentration might also lower the compound apparent water solubility.
260 261 262 263
Table 2- Chitosan nanoparticles (CH-NP) average size and polydispersity index (PDI) in the presence or not of polysorbate 20 (P20). Error bars correspond to standard deviations of three measurements. System CH-NP without P20 CH-NP with P20 CH-5-NFB-NP with P20
Average size (d, nm) 209.2 ± 1.7 320.2 ± 3.1 321.5 ± 3.2
PDI 0.157 ± 0.02 0.202 ± 0.01 0.180 ± 0.01
264 265
Our results show the influence of salt concentration in chitosan nanoparticles size, PDI
266
and zeta potential. More important, we show that for systems with nonionic surfactants, salt
267
addition is deleterious, especially if the drug is preferentially incorporate in the surfactant
268
micelles. Also, we show that chitosan nanoparticles can be an alternative for hydrophobic drugs
269
incorporation when the drug is initially solubilized in surfactant micelles.
10
270
3.2. Thermal analyses
271
The DSC analyses were performed with Ch-5-NFB-NP at different concentrations of
272
saccharose, lactose and glycine (0, 2.5, 5 and 10%) to determine the glass transition temperature
273
(Tg’) and eutectic temperature (Teut) (Table 3). The lyophilization with optical microscopy was
274
performed with the samples to determine the collapse temperature (Tcol). The Tg’ is defined by
275
the transition of viscous state (bloody) to stiff state of amorphous solid in freezing process, with
276
viscosity increase. The Teut is the collapse temperature of crystalline compounds between
277
freezing and melting point of these compounds. The Tcol is the temperature that the frozen
278
structure does not support itself in the primary drying because of high water mobility [21].
279
Determination of Tg’, Teut and Tcol is very important for freeze-drying processes because
280
the amorphous or crystalline solid have to be in stiff state during primary freeze-drying. If the
281
primary drying occurs above Tg’, Teut and Tcol, the water is sufficient free in interstitial region
282
and can no longer keep its original configuration, leading to a collapse duo to low matrix
283
viscosity above these temperatures. So, the freeze-drying has to occur below Tg’, Teut and Tcol.
284
According to DSC analyses, the sample CH-5-NFB-NP 2.5% glycine did not present a Tg’. The
285
samples CH-NP, Ch-5-NFB-NP 10% lactose, CH-5-NFB-NP 10% glycine and all concentrations
286
of CH-5-NFB-NP saccharose did not present Teut. The Teut demonstrated in CH-5-NFB-NP
287
evidence the crystalline character of 5-NFB in CH-5-NFB-NP.
288 289
The Tcol was determined base optical cryomicroscopy images (Figure 2) to preview the minimal temperature that freeze-drying process have to occur without “cake” collapse.
11
290 291 292 293 294 295 296 297 298 299
Figure 2- Microscope images of lyophilization process of CH-5-NFB-NP without excipients. a) liquid structure; b) frozen structure; c) freeze-drying layer; d) microcollapse; e) macrocollapse; f) total collapse. This experiment was performed with glycine, lactose and saccharose at 2.5, 5 and 10%.
Table 3- Values of Teut, Tg’and Tcol (°C) for CH-5-NFB-NP with lactose, saccharose, glycine and water as excipients. Excipient
Tcol
-3.4 -2.4 -
-38.8 -39.9 -44.4 -35.0
-35.5 -28.0 -37.3 -26.4
5% 2.5% 10%
-
-35.0 -37.0 -37.0
-26.6 -23.2 -3.9
5% 2.5%
-5.2 -7.8
-24.0 -
-11.3 -38.5
-
1.4
-
-24.3
Saccharose
Glycine
Lactose
Water
300
Tg’
Concentration 10% 5% 2.5% 10%
Teut
12
301
In freeze-drying process, the higher the Tg’ and Tcol’ temperatures the faster and cheaper is
302
the process. Temperatures below -40 °C are not viable, since the process becomes slow and
303
expansive. The higher Tg’ was observed with Ch-5-NFB-NP 5% glycine (Tg’= -24 °C) and the
304
higher Tcol was observed with CH-5-NFB-NP 10% glycine (-3.9 °C).
305
3.3 Freeze-drying
306
In freeze-drying process excipients are usually employed as bulking agents, lyoprotectants
307
and cryoprotectants. Among them, one can cite polysaccharides, saccharides, proteins and
308
aminoacids. The bulking agents are responsible to provide volume to dry matrix, replacing the
309
water and forming a crystalline structure that supports the cake. Additionally, cryoprotectants
310
protect the active agent during freeze-drying process [20]. The primary freeze-drying process
311
occurred with about 20 hours and the cakes were kept under vacuum (Figure 3).
312
1
2
3
4 313 314 315 316 317
Figure 3- Lyophilized cakes of CH-5-NFB-NP with different excipients. Line 1: water; Line 2: glycine at 2.5, 5 and 10%; Line 3: lactose at 2.5, 5 and 10%; Line 4: 2.5, 5 and 10%.
13
318
The glycine cakes were adequate, with no shrinkages and cracks, considered ideal to the
319
formulation preservation, transportation and commercialization. The CH-5-NFB-NP lyophilized
320
cakes with lactose and saccharose were acceptable, but presented shrinkages and cracks, most of
321
them not associated with collapse. These phenomena are related with stress in primary drying, in
322
which the water in the sample leaves the structure causing stress and forming shrinkages and/or
323
cracks. Shrinkages are usually associated to low concentrations of bulking agents and cracks are
324
associated to high concentrations. However, these events are acceptable and do not interfere in
325
product quality, except to possible cake deconfiguration during transportation [22].
326
All cakes were immediately resuspended in ultrapure water and the characteristics of the
327
reconstituted systems are presented in Table 4. Glycine is widely employed as bulking agent in
328
freeze-drying process due to its low Tg’ and Tcol’, resulting in elegant cakes. Additionally,
329
glycine is safe and biocompatible as an excipient. In our case glycine did not result in adequate
330
systems after the nanoparticles reconstitution. The CH-5-NFB-NP with glycine 2.5% and 5%
331
presented aggregates a few minutes after reconstitution, as can be seen for the high PDI values
332
observed for both samples. Only the highest concentration of glycine (10%) was able to stabilize
333
the CH-5-NFB-NP, visually preserving colloidal stability. In neutral pH, glycine is totally ionized
334
and the negative charges of the carboxylic groups can electrostatically interact with the positive
335
nanoparticles surface. As a consequence, glycine may shields the nanoparticles surface resulting
336
in lower zeta potential value, since the amine groups of glycine that will be at the nanoparticles
337
surface are weaker than amine groups of chitosan. At lower zeta potential values, nanoparticles
338
aggregation was observed.
339 340
Table 4- Lyophilized CH-5-NFB-NP characteristics after resuspension. Lyophilized Sample
Average size (d.nm)
PDI
Zeta Potential (mV)
CH-5-NFB-NP
Not resuspend
-
-
Relative Moisture (%) 3.5
CH-5-NFB-NP
607.0 ± 21.5
glycine 2.5%
(aggregates)
0.765 ± 0.19
+12.9 ± 4.75
3.55
CH-5-NFB-NP
352.4 ± 5.23
glycine 5%
(aggregates)
0.440 ± 0.09
+16.4 ± 4.64
2.49
14
CH-5-NFB-NP glycine 10% CH-5-NFB-NP lactose 2.5% CH-5-NFB-NP lactose 5% CH-5-NFB-NP lactose 10% CH-5-NFB-NP saccharose 2.5% CH-5-NFB-NP saccharose 5% CH-5-NFB-NP saccharose 10%
305.1 ± 3.24
0.251 ± 0.021
+20.3 ± 4.31
2.18
295.5 ± 4.21
0.239 ± 0.034
+31.1 ± 5.31
3.67
299.2 ± 2.49
0.228 ± 0.018
+29.7 ± 4.24
2.80
336.7 ± 3.29
0.259 ± 0.016
+20.9 ± 4.68
1.38
317.4 ± 2.23
0.233 ± 0.019
+33.0 ± 6.26
5.41
331.1 ± 4.5
0.229 ± 0.035
+31.8 ± 5.13
3.73
347.1 ± 2.56
0.253 ± 0.024
+22.6 ± 4.39
3.13
341 342
Both saccharose and lactose were able to preserve the nanoparticles characteristics at all
343
concentrations, except the saccharose 2.5% that resulted in a high moisture (5.41 %) for
344
lyophilized products[20]. For these lyoprotectants, the only interactions are the hydrogen bonds
345
among water molecules and the nanoparticles. Based on our results and previous ones by Allison,
346
Brynildsen and Collins et al. (2011)[23], we believe that saccharose at 5 and 10% can be
347
considered the best lyoprotectant since it demonstrated to be useful for bacterial resistance
348
combat, in which the saccharides were responsible to “wake-up” the latent bacteria, facilitating
349
the bactericidal agents activity.
350
3.4 Scanning Electron Microscopy, (SEM)
351
The morphology of CH-NP and Ch-5-NFB-NP were observed by SEM and, based on the
352
images obtained (Figure 4), both CH-NP and Ch-5-NFB-NP can be considered spherical and with
353
an average diameter of 450 nm, what is significantly larger that the values obtained by DLS
354
measurements (320 nm). We believe this difference might result from the staining process and
355
also from some degree of polymer melting during SEM analysis, owing to the increase in sample
15
356
temperature with the laser beam incidence on the nanoparticles. Nonetheless, SEM is important
357
to confirm the nanoparticles shape, whereas DLS is more adequate to estimate the polymer
358
nanoparticles average size.
359 360 361
Figure 4- Scanning electron microscopy images. a) CH-NP (scale bar = 10 µm) and b) CH-5NFB-NP (scale bar = 5 µm).
362 363
3.5. Antimicrobial activity assay
364
The antimicrobial activity of 5-NFB, CH-5-NFB-NP and CH-NP 5-NFB was determined
365
against strains of S. aureus ATCC 29213, hVISA and ORSA by minimal inhibitory concentration
366
(MIC) and results are presented in Table 5.
367 368 369 370 371
372 373
Table 5- Minimal inhibitory concentration of 5-NFB, CH-5-NFB-NP and CH-NP against S. aureus strains ATCC 29213, hVISA and ORSA, considering 5-NFB MIC present into nanoparticles and nanoparticles MIC (Nps). Error bars correspond to standard deviations of three measurements. Minimal Inhibitory Concentration (MIC)
1.4 – 1.5
CH-5-NFB-NP 5-NFB MIC (µg/mL) 0.4 – 0.5
CH-5-NFB-NP Nps MIC (µg/mL) 10.9 – 14.6
CH-NP Nps MIC (µg/mL) 24.3 – 40.4
Hvisa
1.4 – 1.5
0.8 – 0.9
14.6 – 21.9
24.3 – 32.3
ORSA
2.2 – 2.3
0.6 – 0.8
18.3 – 21.9
24.3 – 32.3
S. aureus strain
5-NFB (µg/mL)
ATCC 29213
16
374
According to the results, 5-NFB showed higher activity against ATCC 29213 and hVISA
375
strains and was less active against ORSA. Previously, our group demonstrated that the 5-NFB
376
activity against ATCC 29213 (5.8 µM) is superior to nifuroxazide (16.0 – 4.0 µM) and
377
vancomycin (<20 µM) [3]. The CH-5-NFB-NP activity was 3 times higher than 5-NFB (in
378
relation to CH-5-NFB-NP/ 5-NFB MIC) and 2 times higher than CH-NP (in relation to CH-5-
379
NFB-NP/ Nps MIC) against ATCC 29213; 2 times higher than 5-NFB (in relation to CH-5-NFB-
380
NP/ 5-NFB MIC) and 1.7 times higher than CH-NP (in relation to Ch-5-NFB-NP/ Nps MIC)
381
against hVISA; 3 times higher than 5-NFB (in relation to CH-5-NFB-NP/ 5-NFB MIC) and 1.3
382
times higher than CH-NP against ORSA (in relation to CH-5-NFB-NP/ Nps MIC). Our results
383
point to a synergistic effect of 5-NFB and chitosan nanoparticles since the concentration of
384
compound and chitosan nanoparticles in the CH-5-NFB-NP necessary to inhibit the bacterial
385
growth is lower than 5-NFB and Ch-NP isolated effects. The antibacterial mechanism of 5-NFB
386
is related to reduction of nitro group in toxic radicals that interferes in DNA and RNA cellular
387
synthesis [3]; the antibacterial mechanism of chitosan, on the other hand, is related to its
388
protonated amino groups that may interact with negative charges of bacterial proteins and other
389
cellular constituents [8]. This is very interesting regarding antibacterial resistance, when two or
390
more antibacterial agents used together with distinct mechanisms may decrease the antimicrobial
391
resistance [24]. These results demonstrated that this formulation could be effective to eliminate
392
resistant infections.
393 394
4. CONCLUSIONS
395
In this paper, we present a novel formulation for a promising antibacterial compound, 5-
396
NFB: N’-((5-nitrofuran-2-yl)methylene)-2-benzhydrazide. Since it is a hydrophobic compound,
397
we solubilized it in Polysorbate 20 micelles that were further incorporated in chitosan
398
nanoparticles. The CH5-NFB-NP presented a diameter of 321 nm, PDI of 0.18, ζP +37 mV and
399
an encapsulation efficiency of 44%, resulting in good parameters to topical application and
400
nanoparticles stability. Samples were successfully lyophilized using saccharose or lactose as bulk
401
agents. and SEM analyses showed spherical and regular nanoparticles. The CH-5-NFB-NP
402
presented increased activity in comparison to the free compound and empty chitosan
403
nanoparticles (CH-NP) against all S. aureus strains, including multidrug-resistant strains (hVISA
404
and ORSA). In addition, our results point to a synergic antimicrobial activity between chitosan
17
405
nanoparticles and 5-NFB. Therefore, CH-5-NFB-NP demonstrated to be a promising alternative
406
for the treatment of S. aureus infections, especially in skin burned areas. On a broader
407
perspective, several papers present interesting results in the design and preparation of chitosan
408
nanoparticles. However, they are usually designed for the incorporation of drugs based on
409
entrapment and/or electrostatic interactions of the drugs with the cross-linked chitosan net. This
410
is the first time a complex system based on the encapsulation of a hydrophobic drug into
411
Polysorbate micelles with further entrapment in chitosan nanoparticles is described. It opens the
412
possibility of other hydrophobic drugs incorporation into similar systems.
413
Acknowledgments
414
The authors acknowledge the financial support of the Coordination for the Improvement of
415
Higher Education Personnel - CAPES (Process 001) and the National Council for Scientific
416
and Technological Development-CNPQ.
417
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Highlights ““Chitosan nanoparticles for the delivery of a new compound active against multidrugresistant Staphylococcus aureus” Laura Freitas de Andrade, Alexsandra Conceição Apolinário, Carlota de Oliveira RangelYagui, Marco Antonio Stephano, Leoberto Costa Tavares. • • •
The efficient loading of lipophilic compound 5-NFB into chitosan nanoparticles; Efficient lyophilization of Ch-5-NFB-NP with lactose and sacharose as bulk agents; Great activity of Ch-5-NFB-NP against multidrug resistant Staphylococcus aureus.
Conflict of Interest Statement
“Chitosan nanoparticles for the delivery of a new compound active against multidrugresistant Staphylococcus aureus” The authors whose names are listed immediately below certify that they have NO affiliations with or involvement in any organization or entity with any financial interest (such as honoraria; educational grants; participation in speakers’ bureaus; membership, employment, consultancies, stock ownership, or other equity interest; and expert testimony or patent-licensing arrangements), or non-financial interest (such as personal or professional relationships, affiliations, knowledge or beliefs) in the subject matter or materials discussed in this manuscript. 1. 2. 3. 4. 5.
Laura Freitas de Andrade Alexsandra Conceição Apolinário Carlota de Oliveira Rangel-Yagui Marco Antonio Stephano Leoberto Costa Tavares