D E N T A L M A T E R I A L S 3 3 ( 2 0 1 7 ) e53e61

Available online at www.sciencedirect.com

ScienceDirect

j o u rn al homepage:www.intl.elsevierhealth.com/journals/dema

New denture adhesive containing miconazole nitrate

polymeric microparticles: Antifungal, adhesive force and
toxicity properties
a,
Andrs Felipe Cartagena , Lus Antonio Esmerino , Rogerio Polak-Junior ,
b c
a c b
Sibelli Olivieri Parreiras , Milton Domingos Michl , Paulo Vitor Farago ,
a
Nara Hellen Campanha
a
Department of Dentistry, State University of Ponta Grossa, Ponta Grossa, Brazil
b
Department of Pharmaceutical Sciences, State University of Ponta Grossa, Ponta Grossa, Brazil
c
Department of Materials Science and Engineering, State University of Ponta Grossa, Ponta Grossa, Brazil

AR T I C L E I N F O A BS T R A C T

Article history: Objective. The purpose of this study was to develop a new oral drug delivery system by incor-porating
Received 24 April 2016 polymeric miconazole nitrate (MN) microparticles on an experimental antifungal denture adhesive (DA).
Received in revised form 14
September 2016 Methods. Spray drying Eudragit L-100 (E) and Gantrez MS-955 (G) MN-microparticles were incorporated in
Accepted 27 September 2016 DA. DAE1, DAG1, DAEG1, DAE2, DAG2, DAEG2 groups were obtained from the combination of
polymers used in MN-microparticles (E, G and EG) and concentration of MN into DA (1, for 1% and 2, for
2%). DA with 2% pure MN (DAM) and DA without micropar-ticles or drug (DACT) were both control
Keywords: groups. All groups were evaluated to determine microbiological assay, adhesive force and toxicity. Minimum
Complete denture inhibitory concentration (MIC) against Candida albicans was performed by broth micro-dilution and agar

Denture adhesives dilution methods in extract of DAs and conventional gel form (Daktarin ). Adhesive load testing was made
Denture stomatitis between acrylic resin samples on a universal testing machine after immersion in water. The toxicity of several
Miconazole nitrate dilutions of DAs was performed with Artemia salina bioassay after 24 and 48 h. Data of adhesive force were
Microparticles drug delivery evaluated with two-way ANOVA and Bonferroni tests (a = 0.05).

Results. The concentration required to kill 50% (LC50) was determined using the Provit anal-ysis. DA with
polymeric microparticles and pure drug presented MIC between 1.255 mg/mL similar to MIC values of
DAM. DAEG2, DAEG1, DAG20 showed the most actives against C. albicans. The best adhesive properties
were exhibited by DAEG2, consisting of high initial adhesive force which was maintained for up to 6 h. The
extracts of all DA presented low or not toxicity at 24 and 48 h.

Significance. DA containing 2% of MN loaded in microparticles made by Gantrez MS-955 alone or combined

with Eudragit L-100 produce effective antifungal activity, good adhesive force,

Corresponding author at: Universidade Estadual de Ponta GrossaPs-graduac o em Odontologia, Rua Carlos Cavalcanti, 4748, Bloco M, Sala 64A
Uvaranas, Ponta Grossa, Paran 84030-900, Brazil.
E-mail address: afelipe87@hotmail.com (A.F. Cartagena).
http://dx.doi.org/10.1016/j.dental.2016.09.039
0109-5641/ 2016 The Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.
e54 D E N T A L M A T E R I A L S 3 3 ( 2 0 1 7 ) e53e61

and no toxicity effect being a promising therapeutics for removable denture wearers affected by denture
stomatitis.
2016 The Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

contact time in the target area [27,28] leading to high initial concentrations
1. Introduction
of MN, followed by low levels, requiring multi-ple administrations each
day [22]. Furthermore, MN has very low aqueous solubility leading to
Denture stomatitis (DS) is the erythematous inflammatory condition more
erratic and unpredictable bioavailability [29]. Drug delivery devices
frequently found in patients with removable dentures [13]. When
containing MN have been developed for topical treatment of oral mycoses
affected patients have symptoms, they often complain of a burning
in for-mulations such as gel [29], chewing gum [30], bioadhesive films
sensation, unpleasant taste and discomfort. In most cases, DS can occur
[31] and buccal tablets [32].
without any symp-toms, and individuals without knowledge of the disease
are commonly found. Classically, estimation of clinical disease severity
In order to increase bioavailability and to achieve the ther-apeutic
has been based on different clinical manifestations categorized according
goals, spray dried polymeric microparticles of MN presenting
to the severity of inflammation [4]. Inde-pendent of the contribution
improvements in drug solubility and antifungal activity against C. albicans
factors like age, systemic disease, smoking, wearing complete dentures
have been developed [33]. These microparticles were formulated using
during sleep, reduced salivary flow, trauma caused by the lack of retention
Eudragit L-100 polymer, which has a favorable release profile on oral pH
and stabil-ity of the prosthesis, the fungus Candida albicans is considered
(pH-sensitive) [34,35] and the muco-adhesive polymer Gantrez MS-955,
as the primary etiological factor of DS. Despite the prevalence of this
rec-ognized to extend the residence time of drugs on several membranes
microorganism [5,6], non-albicans species (e.g., Can-dida glabrata,
[36,37].
Candida tropicalis, Candida guilliermondii, Candida dubliniensis,
Candida parapsilosis and Candida krusei) [610] can also be responsible
The purpose of the present study was to make preliminary in vitro tests
for the disease, among other microorgan-isms [11].
(antimicrobial against C. albicans, adhesive force calculation and Artemia
salina toxicity bioassay) of drug deliv-ery systems consisting of
antifungal-DA formulations aiming at DS treatment. These systems were
obtained by the incor-poration of pH-sensitive (Eudragit L-100) and
It has been reported resistance of Candida species to con-ventional
muco-adhesive (Gantrez MS-955) polymeric MN-microparticles on the
anti-fungal agents and ineffective therapies. The strategies to solve this
compo-sition of an experimental DA.
problem, specifically in DS, has focused on oral hygienic education [12],
phytotherapy [13], controlling the factors that may cause trauma to the
mucosa and search-ing developing new pharmaceutical formulations [14
16]. 2. Material and methods
The use of denture adhesives (DA) for the improvement on retention
and stability of removable dentures it is very common [17]. These 2.1. Experimental design
materials are available in cream, strip, powder and cushion formulations
and can be classified as muco-adhesive compositions. Usually, DA cream The influence of incorporated spray dried microparticles of MN made with
formula-tion is made of a mixture of water-soluble polymers that provide the polymers Eudragit L-100 (E), Gantrez MS-955 (G) or the combination
muco-adhesive characteristics and base components that facilitate the of both (EG) and drug concentrations (10 and 20%) in an experimental
placement of it [18,19]. Muco-adhesion is a promising strategy for cream den-ture adhesive (DA) was studied. These microparticles were
adequate and prolonging drug release of the conventional oral medications developed and proven adequate about drug loading and encapsulation
[20,21]. Muco-adhesive formulations can be attached to the mucosa for efficiency [33]. In the present work, the experi-mental DA was prepared
extended peri-ods of time, allowing an increase of drug bioavailability by a mechanical process, mixing the active water-soluble polymers and
[22]. They have offered better control of antifungal activity as com-pared base components (Table 1). Microbiological activity against C. albicans,
to a marketed gel formulation [15]. adhesive force (ten-sile test) and toxicity (against A. salina) were
investigated to study the DA added or not with the microparticles. A com-

monly used MN gel form Daktarin was used as control when necessary.
Some authors have reported a retentive effect of DA of 68 h in
denture wearers [17]. This period of use and the muco-adhesive properties
of DA could be employed for the drug release of antimicrobial agents
such as MN in the therapy of DS. Few studies have investigated this
option [23,24], although some DAs have already showed antimicrobial 2.2. Spray-drying polymeric microparticles of MN
efficacy against oral microorganisms associated to oral malodor [25].
Weighted amounts of polymers (E, G, or EG) and MN was dis-solved in
Miconazole nitrate (MN) is one of the first line broad-spectrum ethanol or/and water. The solution obtained were then mixed and magnetic
triazole antifungal agent used [26] to treat superficial mucosal candidiasis. stirred for 6 h. Spray-drying was per-formed using a MSD-0.5 mini spray
MN oral gel is the more widely available formulation worldwide. dryer (Labmaq, Ribeiro Preto, Brazil) with a standard 0.5 mm nozzle.
Traditional oral gel has short Outlet tempera-
 D E N T A L M A T E R I A L S 3 3 ( 2 0 1 7 ) e53e61 e55

Table 1 Components used in denture adhesive formulation.

Material Manufacturer Lot #. wt%

Gantrez MS-955 ISP, Wilmington, Delaware, USA CC2305825 37.5

Sodium carboxymethyl cellulose (CMC) ISP, Wilmington, Delaware, USA 70028 15.5

Liquid parafin Ind. e Farm. LIFAR LTDA, Porto Alegre, Rio Grande do Sul, Brazil L32456 10.4

White petrolatum Ind. e Farm. LIFAR LTDA, Porto Alegre, Rio Grande do Sul, Brazil AC8763 28.5

Versagel MN Penreco, Karns City, Pensilvania, EUA K1002 5
Colloidal Silica Evonik Industries, Essen, Germany 0288 3

BHT Mapric, So Paulo, So Paulo, Brazil 329850 0.05

Nipazol (propylparaben) Aboisa, Santa Ceclia, So Paulo, Brazil R24591 0.05

Fig. 1 Schematic showing the elaboration of the adhesive extract and the microbiological tests for MIC.

addition of any microparticles (DACT) were used as controls. All along

ture was maintained at 60 C, flow rate was kept at 56 mL/min,
compressed spray airflow was maintained at 40 L/min and air pressure the text the above mentioned formulations of DA were tested by different
3 methods.
was kept at 1.5 m /min. The obtained microparti-cles were separated in a
cyclone separator and settled into a collector protected from light.
2.4. Microbiological assays

2.3. Preparation of denture adhesives
A DA was developed from two types of synthetic water-soluble polymers
(sodium carboxymethyl cellulose and Gantrez MS-955) and base
materials. Details and percentages of materials used in the DA
formulation can be seen in Table 1. All the for-mulation components were
adequately weighed and mixed by a mechanical vacuum mixer at 2400
rpm for 20 min. Ten per-cent (w/w) of MN microparticles were
incorporated on the DA and mixed, to achieve final concentrations of 1%
and 2% of MN. Then, six DA were obtained from the combination of
polymers used in the formulation of microparticles and the final con-
centration of MN (DAE1, DAG1, DAEG1, DAE2, DAG2, DAEG2). DA
formulations with 2% of pure MN (DAMN) and without the
Extracts of all DA formulations were prepared in Mueller-Hinton broth at
2% w/v [3841] and at MN theoretical concentration from 0.31240
mg/mL. MICs were determined by both a broth micro-dilution method
based on the NCCLS M27-A [42] with modification and an agar dilution
method based on the NCCLS M27-P [43] (Fig. 1). Cells of C. albicans
ATCC 10231 were resuspended in saline, and suspension adjusted to 0.5
McFarland standard and then diluted 10-fold with saline to give a yeast
5 5
suspension of 0.5 10 to 2.5 10 CFU per mL.
The agar dilution test was performed with Mueller-Hinton agar 2%
glucose. The agar medium (18 mL) was poured into Petri dishes
containing 2 mL of serial dilution of DA in broth medium and was
solidified. The suspension of C. albicans was loaded on a sterile cotton
swab and plated by stirring the swab
e56 D E N T A L M A T E R I A L S 3 3 ( 2 0 1 7 ) e53e61
Fig. 2 Acrylic resin cylinders aligned and placed in a tailor-
developed device in universal testing machine.
over the entire surface. For the broth dilution assay, it was used a
modified CLSI M27-A3 microdilution method. Aliquots of 180 mL of the
C. albicans suspensions in Mueller-Hinton broth were inoculated into
microtiter plate wells containing 20 mL of DAs dilutions.
Registers of Petri dishes (from agar dilution test) and microplates
(from broth microdilution test) were made after incubated at 37 C for 24
h. MICs were defined as the lowest concentration that suppressed any
visible growth, or causing almost complete inhibition of growth for both
tests. All tests were carried in triplicate.
2.5. Adhesive force measurement
The measurement of adhesive force was performed between two cylinders
of a denture base polymethylmethacrylate resin (Vipi wave, Pirassununga,
SP, Brazil). The cylinders were microwave heat-processed on dimensions
of 25 mm (diame-ter) 35 mm (height) and aligned in a tailor-developed
device for a AG-I Shimadzu Autograph (Shimadzu, Kyoto, Japan) test
machine (Fig. 2). The resin test surface was finished with 600-grit silicon
carbide paper. Amounts of formulations of the DA (0.3 g) were weighted
and immersed in distilled water at 37 C for 0.5, 1, 3 and 6 h. Thus, DA
was applied on the top of cylinders assembled in the test machine and 12
N compres-sion force was initially applied for 30 s to simulate a gentle
occlusal force [44,45]. Finally, the tensile test was performed at crosshead
speed of 1 mm/min and the maximum force before failure was calculated
(N). Each test was repeated 10 times. For
Fig. 3 Adhesive force mean values (N) as determined by tensile test
for all formulations of denture adhesives investigated in the present
work as a function of time.
all tests, the surface of acrylic resin was cleaning with neu-tral soap,
washed with distilled water and dried with paper towel. After this, new
portions of DA were applied on the top of the cylinders. Data were
submitted to two-way analysis of variance (ANOVA) and Bonferroni tests
(a = 0.05).
2.6. Toxicity bioassay
A. salina bioassay, according to the methodology of Meyer et al. [46], was
used for toxicity determination of DA extracts. Brine shrimp eggs were
placed in saline (3.8% w/v sea salt in dis-tilled water) and incubated at 24
28 C, pH between 78 and constant illumination. Eggs hatched and
matured within 48 h providing large number of larvae (nauplii). Ten
nauplii were placed in vials containing 5 mL of seawater and increasing
concentrations of DA extracts (251000 mL/mL). Serial dilutions were
prepared in order to reach the chosen concentrations in the final volume (5
mL). Controls were made in vials contain-ing 5 mL of saline and 0.1%
potassium dichromate at the same dilution concentration of extracts. The
vials were maintained under illumination. All experiments were carried
out in trip-licate. Alive nauplii were recorded after 24 and 48 h using a
stereomicroscope. The data were corrected using Abbotts for-mula as
follows: %deaths = [(Test-control)/control 100] [47]. The concentration
required to kill 50% (LC50 ) was determined using the Provit analysis.
3. Results
3.1. Results of microbiological assays
The MIC values of the seven tested extracts of DA against C. albicans
ATCC 10,231 at 24 h are summarized in Table 2. The results confirmed
the antifungal activity of extracts of DA that contain MN or MN-
microparticles for both methods employed. The range of MICs was 1.255
mg/mL. The most active extracts against C. albicans, with MIC = 1.25
mg/mL, were DAEG1, DAG2 and DAEG2 (microdilution) and DAEG1,
DAE2, DAG2 and DAEG2 (agar dilution). Extracts of DAMN, DAE1 and
 D E N T A L M A T E R I A L S 3 3 ( 2 0 1 7 ) e53e61 e57

Table 2 Minimum inhibitory concentrations of denture adhesives at 24 h.

Range (mg/mL) of MN Denture adhesive extract Broth microdilution (mg/mL) Agar dilution (mg/mL)
0.31240 DACT
DAMN 2.5 5
DAE1 2.5 2.5
DAG1 2.5 2.5
DAEG1 1.25 1.25
DAE2 2.5 1.25
DAG2 1.25 1.25
DAEG2 1.25 1.25

Daktarin 2.5 2.5
DACT: pure experimental denture adhesive, i.e., without miconazole; DAMN: experimental denture adhesive added by 2% miconazole nitrate; DAE1: experimental
denture adhesive added by 1% miconazole nitrate encapsulated in Eudragit L-100 microparticles; DAG1: experimental denture adhesive added by 1% miconazole nitrate
encapsulated in Gantrez MS-955 microparticles; DAEG1: experimental denture adhesive added by 1% miconazole nitrate encapsulated in a mixture of Eudragit L-100 and
Gantrez MS-955 microparticles; DAE2: experimental denture adhesive added by 2% miconazole nitrate encapsulated in Eudragit L-100 microparticles; DAG2:
experimental denture adhesive added by 2% miconazole nitrate encapsulated in Gantrez MS-955 microparticles; and DAEG2: experimental denture adhesive added by 2%
miconazole nitrate encapsulated in a mixture of Eudragit L-100 and Gantrez MS-955 microparticles.

DAG1 were very similar against C. albicans to conventional MN gel form

4. Discussion
(Daktarin ).

Prior to clinical tests and endorsement, it seems meaningful to conduct

3.2. Results of adhesive force measurement
The ANOVA results (Table 3) indicate significant differences among the
tested materials and reveal significant effects of immersion time on
adhesive force (p < 0.000). Fig. 3 shows the variation on adhesive force of
the tested materials accord-ing to immersion time. Overall, DA
formulations significantly increase the adhesive force up in the intervals
up to 3-h immersion time and then significantly decrease it (p < 0.000).
Fig. 4 shows de adhesive force of each DA formulations, inde-pendently
of time. The addition of pure MN in the adhesive formulation exhibited
statistically similar values of adhe-sive force along the evaluated times.
Overall, formulations containing only Eudragit L-100 polymer have
shown lower (p < 0.000) adhesive force mean values as compared to
control (DACT). Fig. 5 depicts mean values of each combination of DA
formulations and immersion time. It can be seen a tendency of increased
adhesive force up to 3 h and then decrease. In addition, formulations of
DA containing Gantrez MS-955 poly-mer showed a good behavior
maintaining their adhesive force along the time.
3.3. Results of bioassay toxicity
The results of LC50 values of DA extracts are shown in Table 4. All the
tested extracts showed that the concentration was directly correlated to the
death of brine shrimps. Extracts with microparticles or pure MN were
more toxic in comparison to the control formulation (DACT). LC 50
values ranged from 349.53 to 931.00 mg/mL, with DAE2 having the
lowest value (most toxic); this was followed by DAG2 and DAEG2.
Extracts of DA containing MN microparticles at 10% were shown to be
less toxic as compared to extracts of DA with MN at 20%. Toxi-city
assays revealed nine extracts of DA with low or no toxicity at 24 and 48 h
preliminary in vitro antifungal, adhesive force and toxicity tests for a new
approach to DE treatment. The results of this study show that the
following formulations of denture adhesives (DA): DAEG1, DAG2 and
DAEG2 showed good per-formance in the tests carried out. These results
may suggest that the use of MN containing microparticles, in particular
with Gantrez MS-955 polymer, could be a promising alterna-tive to be
added to the DA formulations providing effective antifungal therapy,
thereby avoiding the known disadvantages of conventional forms of MN.
The incorporation of antifungal agents and other tech-nologies in
materials for denture base [48], and, liners [49] and tissue conditioners
[50] have a great potential for inhibiting microorganisms. In this study, we
have attempted incorporating various formulations and concentration of
MN-microparticles on DA to achieve efficient antifungal activity, without
impairment on adhesive strength or increase toxicity.
As it can be seen from Table 2, the MIC of MN added to DA as
determined by broth microdiluiton and agar dilu-tion methods range from
1.25 to 5 mg/mL. These results are in accordance with the values reported
by previous stud-ies for MN in gel form (110 mg/mL) [14]. Effective
release of MN and MN-microparticles in microbiological tests and
compatibility to load anti-fungal agent in DA was demon-strated.
Meanwhile, MICs for some formulations loaded with MN-microparticles
(DAEG1, DAG2, DAEG2) presented better antimicrobial effectiveness
when compared to the DA formu-lation in which the pure MN was added.
These results can be explained by the improved solubility of the MN
obtained after the microencapsulation process [33,51]. Some authors have
reported problems with topical formulations of MN, and therefore,
compromised bioavailability and microbiological effectiveness
[14,16,52]. However, the incorporation of the MN microparticles in
matrix of DA did not impair its microbiolog-ical efficacy. In the present
work, MICs were reported as the minimal concentration able to inhibit
any visible growth at 24 h. Plating of the wells content was not
performed. Maybe

(LC50 > 250 mg/mL).

e58 D E N T A L M A T E R I A L S 3 3 ( 2 0 1 7 ) e53e61

Table 3 Summary table analysis showing sources of variability and associated degrees of freedom in analytic model.
Source Contrasts Type III sum of squares df Mean square F P
a
Corrected model 2,100,875 31 67.77 20.987 <0.000
Intercept 26,727.866 1 26,727.866 8277.155 <0.000

Time 1028.595 3 342.865 106.179 <0.000

1 vs 0.5 <0.000
3 vs 0.5 <0.000
6 vs 0.5 <0.000
DA 331.176 7 47.311 14.651 <0.000
DAMN vs DACT 1.000
DAE1 vs DACT 0.002
DAG1 vs DACT 0.034
DAEG1 vs DACT 1.000
DAE2 vs DACT <0.000
DAG2 vs DACT 1.000
DAEG2 vs DACT 0.046

Time DA 741.104 21 35.291 10.929 <0.000

Error 413.326 128 3.229
Total 29,242.068 160
Corrected total 2514.202 159

DACT: pure experimental denture adhesive, i.e., without miconazole; DAMN: experimental denture adhesive added by 2% miconazole nitrate; DAE1: experimental denture
adhesive added by 1% miconazole nitrate encapsulated in Eudragit L-100 microparticles; DAG1: experimental denture adhesive added by 1% miconazole nitrate
encapsulated in Gantrez MS-955 microparticles; DAEG1: experimental denture adhesive added by 1% miconazole nitrate encapsulated in a mixture of Eudragit L-100 and
Gantrez MS-955 microparticles; DAE2: experimental denture adhesive added by 2% miconazole nitrate encapsulated in Eudragit L-100 microparticles; DAG2:
experimental denture adhesive added by 2% miconazole nitrate encapsulated in Gantrez MS-955 microparticles; and DAEG2: experimental denture adhesive added by 2%
miconazole nitrate encapsulated in a mixture of Eudragit L-100 and Gantrez MS-955 microparticles; df: degrees of freedom.

Fig. 4 Adhesive force mean values (N) as determined by tensile test for each formulation of denture adhesives investigated in the present work
independent of time. DACT: pure experimental denture adhesive, i.e., without miconazole; DAMN: experimental denture adhesive added by 2%
miconazole nitrate; DAE1: experimental denture adhesive added by 1% miconazole nitrate encapsulated in Eudragit L-100 microparticles; DAG1:
experimental denture adhesive added by 1% miconazole nitrate encapsulated in Gantrez MS-955 microparticles; DAEG1: experimental denture adhesive
added by 1% miconazole nitrate encapsulated in a mixture of Eudragit L-100 and Gantrez MS-955 microparticles; DAE2: experimental denture adhesive
added by 2% miconazole nitrate encapsulated in Eudragit L-100 microparticles; DAG2: experimental denture adhesive added by 2% miconazole nitrate
encapsulated in Gantrez MS-955 microparticles; and DAEG2: experimental denture adhesive added by 2% miconazole nitrate encapsulated in a mixture
of Eudragit L-100 and Gantrez MS-955 microparticles.
 D E N T A L M A T E R I A L S 3 3 ( 2 0 1 7 ) e53e61 e59

Table 4 Toxicity of denture adhesive extracts against nauplii of Artemia salina.

Adhesive extract 24 h 48 h

LC50 mg/mL Toxicity classification LC50 mg/mL Toxicity classification

DACT >1000 NT or low 931.00 NT or low
DAMN >1000 NT or low 645.07 NT or low
DAE1 >1000 NT or low 809.19 NT or low
DAG1 >1000 NT or low 545.29 NT or low
DAEG1 >1000 NT or low 821.54 NT or low
DAE2 >1000 NT or low 349.53 NT or low
DAG2 >1000 NT or low 353.71 NT or low
DAEG2 >1000 NT or low 438.96 NT or low

NT = non toxic; DACT: pure experimental denture adhesive, i.e., without miconazole; DAMN: experimental denture adhesive added by 2% miconazole nitrate; DAE1:
experimental denture adhesive added by 1% miconazole nitrate encapsulated in Eudragit L-100 microparticles; DAG1: experimental denture adhesive added by 1%
miconazole nitrate encapsulated in Gantrez MS-955 microparticles; DAEG1: experimental denture adhesive added by 1% miconazole nitrate encapsulated in a mixture of
Eudragit L-100 and Gantrez MS-955 microparticles; DAE2: experimental denture adhesive added by 2% miconazole nitrate encapsulated in Eudragit L-100 microparticles;
DAG2: experimental denture adhesive added by 2% miconazole nitrate encapsulated in Gantrez MS-955 microparticles; and DAEG2: experimental denture adhesive added
by 2% miconazole nitrate encapsulated in a mixture of Eudragit L-100 and Gantrez MS-955 microparticles.

DACT MN-microparticles in the treatment of DS would be feasible approach.

21 DAMN
DAE1 However, this study also found that most formulations tended to lose
DAG1
19 their adhesive efficacy as time progressed. These data agree with the
DAEG1
DAE2 results of other clinical and labo-ratorial studies reporting the loss of
DAG2
17 gradually retention of DA [18,19,54]. This can be explained by the
DAEG2
composition of mate-rials. The active ingredients of the experimental DA
Adhesive Force (N)

15 proposed consisted of CMC and Gantrez MS-955 (Table 1), both water-
soluble polymers with different solubility depending on time. Between the
13 two polymers, the CMC has the scope to pro-vide a strong initial adhesion
force, and Gantrez would have a part in the late material adherence due to
11 present lower solubility in comparison to the other polymer [18,19].
There-fore, it could be said that the incorporation of microparticles
containing the Gantrez MS-955 polymer effectively influenced the
9
maintenance of the adhesive force of the formulation DA as seen in the
results (Table 3). The adhesive force exhibited by the formulations
7
containing Gantrez MS-955 microparti-cles was as expected. Bioadhesive
microparticles in DA may have improved adhesive capacity as the
5
solubilization of the polymer occurred. With the incorporation of Gantrez
0.5 1 3 6
Time (h) MS-955 microparticles to the AD, additional active component was
available in the hydrated gel matrix formed by hydrophilic and
Fig. 5 Adhesive force (N) means as determined by tensile test of 6 hydrophobic domains. The active role of Gantrez MS-955 polymer in the
denture adhesives over time intervals up to 6 h. late adhesion [19] may have increase the chem-ical interaction at the
DA/PMMA interface and fibril formation [55] maintaining the adhesive
values when compared to the control formulation. As related as DA
containing Eudragit L-100, microparticles promoted some decrease in
any growth would be observed after plating, indicating a bac-teriostatic their adhesive force along the time, thus it is clear the loss of adhesive
effect and not a bactericidal effect. This should be further investigated. force when the ratio of active components is modified.

Muco-adhesion has shown prolonging the residence time of drugs

through various mucosal routes in drug delivery applications [15,21,22].
In the present study, the adhesive force of the experimental formulations
used was maintained for approximately 3 h and in some cases for 6 h.
This result may indicate a better approach in comparison to the classical
non muco-adhesive formulations of oral antifungals such as in the gel
form. In practice, the contact of gel form of MN in the affected area is lost
after a few minutes of removable prosthe-sis use at normal functions of
oral cavity, leading to several times a day (6 times) applications and the
constant inges-tion of formulation [53]. Thus, the use of the DA loaded
with
Although there is a standard test recommended by ISO 10873 [56],
some authors have used other methods for evaluat-ing the adhesive
strength of AD [55,57,58] and muco-adhesive drug delivery systems [59].
The test used here, as suggest by Zhao et al. [54] has the advantage of be
simple not requiring special equipment for accomplishment. Acrylic resin
cylin-ders are easily processed and its positioning in the testing machine is
simple. Nevertheless, this method and the method
e60 D E N T A L M A T E R I A L S 3 3 ( 2 0 1 7 ) e53e61
proposed by ISO 10873 does not consider factors that can influence the
results, such as, presence of natural saliva, keranitized mucosa and
intaglio surface. Finally, it is com-mon in all methodologies the difficulty
in simulating the oral environment of denture users, although, these tests
should be performed to evaluate and compare their effectiveness before
clinical trials were conducted.
In this study the method of choice for toxicological test-ing is the
brine shrimp lethality. The brine shrimp lethality assay is established as a
rapid, lower cost, and convenient method to predict the toxic effects of
dental materials and it has been used routinely in the primary screening
providing indication of possible cytotoxic properties of the test materials
[60,61]. Some studies have been found using this methodol-ogy in
assessing the toxicity in dental materials [47,61]. The present
experimental formulations have demonstrated lower or no toxicity with A.
salina test (CL50 349.53931.00 mg/mL). This result can be compared
with some available cytotoxi-city studies that have reported some degree
of cytotoxicity between moderate and slightly in commercial DA with a
similar composition to the formulations proposed in this study [54,62,63].
However, it was confirmed by the results of this study increased toxicity
dependent on concentration of extract and evaluation time of assay. On
the other hand, evi-dence anti-fungal drug toxicity has been showed, when
MN or MN-microparticles were added to the DA formulations.
In the current study, DA loaded with MN have showed good results in
the initial tests of microbiological efficient, adhe-sive retention and
toxicity. However, other in vitro studies are required to obtain more
information about the behavior of the proposed formulation of DA, such
as assessments in simple or compounds biofilms of Candida species,
cytotoxicity in fibroblasts or epithelial cells, muco-adhesive test and drug
release. Furthermore, in-vitro studies cannot be extrapolated clinically, for
this reason, studies in vivo should be conducted to provide safety and
clinical performance.
5. Conclusions
The incorporation of polymeric MN-microparticles in DA resulted in
effective strategy as dosage form for the MN without changing their
antifungal effect, adhesive force and toxicity properties. From the
preliminary results, some of the tested antifungal loaded DA have a great
potential for treat-ment of DS, provided that it is further tested.
REFERENCES
[1] Barbeau J, Seguin J, Goulet JP, de Koninck L, Avon SL, Lalonde B, et
al. Reassessing the presence of Candida albicans in denture-related
stomatitis. Oral Surg Oral Med Oral Pathol Oral Radiol Endod
2003;95:519.
[2] Emami E, Taraf H, de Grandmont P, Gauthier G, de Koninck L,
Lamarche C, et al. The association of denture stomatitis and partial
removable dental prostheses: a systematic review. Int J Prosthodont
2012;25:1139.
[3] Gendreau L, Loewy ZG. Epidemiology and etiology of denture
stomatitis. J Prosthodont 2011;20:25160.
[4] Newton AV. Denture sore mouth. A possible etiology. Br Dent J
1962:35760.
[5] Rodrigues JA, Hofling JF, Tavares FC, Duarte KM, Goncalves RB,
Azevedo RA. Evaluation of biochemical and serological methods to
identify and clustering yeast cells of oral Candida species by
CHROMagar test, SDS-PAGE and ELISA. Braz J Biol 2004;64:31726.
[6] Sullivan DJ, Moran G, Donnelly S, Gee S, Pinjon E, McCartan B, et al.
Candida dubliniensis: an update. Rev Iberoam Micol 1999;16:726.
[7] Coleman DC, Bennett DE, Sullivan DJ, Gallagher PJ, Henman MC,
Shanley DB, et al. Oral Candida in HIV infection and AIDS: new
perspectives/new approaches. Crit Rev Microbiol 1993;19:6182.
[8] Gutierrez J, Morales P, Gonzalez MA, Quindos G. Candida
dubliniensis, a new fungal pathogen. J Basic Microbiol
2002;42:20727.
[9] Jabra-Rizk MA, Baqui AA, Kelley JI, Falkler Jr WA, Merz WG,
Meiller TF. Identification of Candida dubliniensis in a prospective
study of patients in the United States. J Clin Microbiol 1999;37:3216.
[10] Vargas KG, Joly S. Carriage frequency, intensity of carriage, and strains
of oral yeast species vary in the progression to oral candidiasis in human
immunodeficiency virus-positive individuals. J Clin Microbiol
2002;40:34150.
[11] Pereira CA, Toledo BC, Santos CT, Pereira Costa AC, Back-Brito
GN, Kaminagakura E, et al. Opportunistic microorganisms in
individuals with lesions of denture stomatitis. Diagn Microbiol
Infect Dis 2013;76:41924.
[12] Ercalik-Yalcinkaya S, Ozcan M. Association between oral mucosal
lesions and hygiene habits in a population of removable prosthesis
wearers. J Prosthodont 2015;24:2718.
[13] Ferreira GL, Perez AL, Rocha IM, Pinheiro MA, de Castro RD, Carlo
HL, et al. Does scientific evidence for the use of natural products in the
treatment of oral candidiasis exist? A systematic review. Evid Based
Complement Alternat Med 2015;2015:147804.
[14] Cardot JM, Chaumont C, Dubray C, Costantini D, Aiache JM.
Comparison of the pharmacokinetics of miconazole after administration
via a bioadhesive slow release tablet and an oral gel to healthy male and
female subjects. Br J Clin Pharmacol 2004;58:34551.
[15] Madgulkar A, Kadam S, Pokharkar V. Development of buccal adhesive
tablet with prolonged antifungal activity: optimization and ex vivo
deposition studies. Indian J Pharm Sci 2009;71:2904.
[16] Rai VK, Yadav NP, Sinha P, Mishra N, Luqman S, Dwivedi H, et al.
Development of cellulosic polymer based gel of novel ternary mixture
of miconazole nitrate for buccal delivery.
Carbohydr Polym 2014;103:12633.
[17] Papadiochou S, Emmanouil I, Papadiochos I. Denture
adhesives: a systematic review. J Prosthet Dent 2015;113:391
7, e2.
[18] Han JM, Hong G, Dilinuer M, Lin H, Zheng G, Wang XZ, et al. The
adhesive strength and initial viscosity of denture adhesives. Acta
Odontol Scand 2014;72:83945.
[19] Han JM, Hong G, Hayashida K, Maeda T, Murata H, Sasaki K.
Influence of composition on the adhesive strength and initial viscosity
of denture adhesives. Dent Mater J 2014;33:98103.
[20] Bartels HA. Bacteriological appraisal of adhesive denture powders. J
Dent Res 1945;24:156.
[21] Shaikh R, Raj Singh TR, Garland MJ, Woolfson AD, Donnelly RF.
Mucoadhesive drug delivery systems. J Pharm Bioallied Sci 2011;3:89
100.
[22] Nafee NA, Ismail FA, Boraie NA, Mortada LM. Mucoadhesive buccal
patches of miconazole nitrate: in vitro/in vivo performance and effect of
ageing. Int J Pharm 2003;264:114.
 D E N T A L M A T E R I A L S 3 3 ( 2 0 1 7 ) e53e61
[23] Scher EA, Ritchie GM, Flowers DJ. Antimycotic denture adhesive in
treatment of denture stomatitis. J Prosthet Dent 1978;40:6227.
[24] Leite AR, Mendoza-Marin DO, Paleari AG, Rodriguez LS, Roccia AA,
Policastro VB, et al. Crossover clinical trial of the influence of the use
of adhesive on biofilm formation. J Prosthet Dent 2014;112:34956.
[25] Polyzois G, Stefaniotis T, Papaparaskevas J, Donta C. Antimicrobial
efficacy of denture adhesives on some oral malodor-related
microbes. Odontology 2013;101:1037.
[26] Bouckaert S, Remon JP. In-vitro bioadhesion of a buccal,
miconazole slow-release tablet. J Pharm Pharmacol 1993;45:504
7.
[27] Gupta A, Garg S, Khar RK. Measurement of bioadhesive strength
of mucoadhesive buccal tablets: design of an in vitro assembly.
Indian Drugs 1993;30:1525.
[28] Vazquez JA, Sobel JD. Miconazole mucoadhesive tablets: a novel
delivery system. Clin Infect Dis 2012;54:14804.
[29] Sawyer PR, Brogden RN, Pinder RM, Speight TM, Avery GS.
Miconazole: a review of its antifungal activity and therapeutic
efficacy. Drugs 1975;9:40623.
[30] Rindum JL, Holmstrup P, Pedersen M, Rassing MR, Stoltze K.
Miconazole chewing gum for treatment of chronic oral candidosis.
Scand J Dent Res 1993;101:38690.
[31] Rassol BKA, Khan SA. In vitro evaluation of miconazole
mucoadhesive buccal films. Int J Appl Pharm 2010;2:236.
[32] Bouckaert S, Schautteet H, Lefebvre RA, Remon JP, van
Clooster R. Comparison of salivary miconazole concentrations
after administration of a bioadhesive slow-release buccal tablet
and an oral gel. Eur J Clin Pharmacol 1992;43:13740.
[33] Cartagena AF, Klein T, Lyra A, Urban AM, Farago PV, Campanha
NH. Development and validation of an RP-HPLC/UV method for
determination of miconazole nitrate in spray-dried polymeric
microparticles. Lat Am J Pharm 2016;35:135460.
[34] Cetin M, Atila A, Kadioglu Y. Formulation and in vitro
characterization of Eudragit(R) L100 and Eudragit(R) L100-PLGA
nanoparticles containing diclofenac sodium. AAPS PharmSciTech
2010;11:12506.
[35] Rizi K, Green RJ, Khutoryanskaya O, Donaldson M, Williams AC.
Mechanisms of burst release from pH-responsive polymeric
microparticles. J Pharm Pharmacol 2011;63:114155.
[36] Kockisch S, Rees GD, Tsibouklis J, Smart JD. Mucoadhesive,
triclosan-loaded polymer microspheres for application to the oral
cavity: preparation and controlled release characteristics. Eur J Pharm
Biopharm 2005;59:20716.
[37] Kockisch S, Rees GD, Young SA, Tsibouklis J, Smart JD. Polymeric
microspheres for drug delivery to the oral cavity: an in vitro evaluation
of mucoadhesive potential. J Pharm Sci 2003;92:161423.
[38] Lee SC, Fung CP, Lee N, See LC, Huang JS, Tsai CJ, et al.
Fluconazole disk diffusion test with methylene blue- and glucose-
enriched Mueller-Hinton agar for determining susceptibility of
Candida species. J Clin Microbiol 2001;39:16157.
[39] Lee SC, Lo HJ, Fung CP, Lee N, See LC. Disk diffusion test and E-test
with enriched Mueller-Hinton agar for determining susceptibility of
Candida species to voriconazole and fluconazole. J Microbiol Immunol
Infect 2009;42:14853.
[40] Pfaller MA, Boyken L, Messer SA, Hollis RJ, Diekema DJ. Stability of
Mueller-Hinton agar supplemented with glucose and methylene blue for
disk diffusion testing of fluconazole and voriconazole. J Clin Microbiol
2004;42:12889.
[41] Rimek D, Fehse B, Gopel P. Evaluation of Mueller-Hinton-agar as a
simple medium for the germ tube production of Candida albicans and
Candida dubliniensis. Mycoses 2008;51:2058.
e61
[42] NCCLS. Reference method for broth dilution antifungal susceptibility
testing of yeasts; approved standard. NCCLS document M27-A.
Wayne, Pa: National Committee for Clinical Laboratory Standards;
1997.
[43] NCCLS. Reference method for broth dilution antifungal
susceptibility testing of yeasts. Proposed standard M27-P. Villanova,
Pa: National Committee for Clinical Laboratory Standards; 1992.
[44] Kore DR, Kattadiyil MT, Hall DB, Bahjri K. In vitro comparison of the
tensile bond strength of denture adhesives on denture bases. J Prosthet
Dent 2013;110:48893.
[45] Haraldson T, Karlsson U, Carlsson GE. Bite force and oral function
in complete denture wearers. J Oral Rehabil 1979;6:418.
[46] Meyer BN, Ferrigni NR, Putnam JE, Jacobsen LB, Nichols DE,
McLaughlin JL. Brine shrimp: a convenient general bioassay for active
plant constituents. Planta Med 1982;45:314.
[47] Pelka M, Danzl C, Distler W, Petschelt A. A new screening test for
toxicity testing of dental materials. J Dent 2000;28:3415.
[48] Wady AF, Machado AL, Zucolotto V, Zamperini CA, Berni E, Vergani
CE. Evaluation of Candida albicans adhesion and biofilm formation on
a denture base acrylic resin containing silver nanoparticles. J Appl
Microbiol 2012;112:116372.
[49] Alcantara CS, Macedo AF, Gurgel BC, Jorge JH, Neppelenbroek KH,
Urban VM. Peel bond strength of resilient liner modified by the addition
of antimicrobial agents to denture base acrylic resin. J Appl Oral Sci
2012;20:60712.
[50] Quinn DM. The effectiveness, in vitro, of miconazole and
ketoconazole combined with tissue conditioners in inhibiting the
growth of Candida albicans. J Oral Rehabil 1985;12:17782.
[51] Davis SS, Illum L. Polymeric microspheres as drug carriers.
Biomaterials 1988;9:1115.
[52] Zhang LW, Fu JY, Hua H, Yan ZM. Efficacy and safety of
miconazole for oral candidiasis: a systematic review and meta-
analysis. Oral Dis 2015;22:18595.
[53] Fothergill AW. Miconazole: a historical perspective. Expert Rev Anti
Infect Ther 2006;4:1715.
[54] Zhao K, Cheng XR, Chao YL, Li ZA, Han GL. Laboratory
evaluation of a new denture adhesive. Dent Mater 2004;20:419
24.
[55] An Y, Li D, Roohpour N, Gautrot JE, Barber AH. Failure
mechanisms in denture adhesives. Dent Mater 2016;32:61523.
[56] ISO. ISO 10873. Dentistry-denture adhesives. Geneva:
International Organization for Standardization; 2010.
[57] DeVengencie J, Ng MC, Ford P, Iacopino AM. In vitro evaluation
of denture adhesives: possible efficacy of complex carbohydrates.
Int J Prosthodont 1997;10:6172.
[58] Koppang R, Berg E, Dahm S, Real C, Floystrand F. A method for
testing denture adhesives. J Prosthet Dent 1995;73:48691.
[59] Chinna Reddy P, Chaitanya KS, Madhusudan Rao Y. A review on
bioadhesive buccal drug delivery systems: current status of formulation
and evaluation methods. Daru 2011;19:385403.
[60] Pelka M, Danzl C, Distler W, Petschelt A. A new screening test for
toxicity testing of dental materials. J Dent 2000;28:3415.
[61] Milhem MM, Al-Hiyasat AS, Darmani H. Toxicity testing of
restorative dental materials using brine shrimp larvae (Artemia
salina). J Appl Oral Sci 2008;16:297301.
[62] de Gomes PS, Figueiral MH, Fernandes MH, Scully C.
Cytotoxicity of denture adhesives. Clin Oral Investig
2011;15:88593.
[63] Al RH, Dahl JE, Morisbak E, Polyzois GL. Irritation and cytotoxic
potential of denture adhesives. Gerodontology 2005;22:17783.