Materials and Design 89 (2016) 294303

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Materials and Design

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In situ forming gel comprising bleached shellac loaded with antimicrobial

drugs for periodontitis treatment
Thawatchai Phaechamud a,, Jongjan Mahadlek b, Tiraniti Chuenbarn a
a
b

Department of Pharmaceutical Technology, Faculty of Pharmacy, Silpakorn University, Nakorn Pathom 73000, Thailand
Pharmaceutical Intelligence Unit Prachote Plengwittaya, Faculty of Pharmacy, Silpakorn University, Nakorn Pathom 73000, Thailand

a r t i c l e

i n f o

Article history:
Received 29 July 2015
Received in revised form 23 September 2015
Accepted 24 September 2015
Available online 8 October 2015
Keywords:
In situ forming gel
Bleached shellac
Antimicrobial

a b s t r a c t
Bleached shellac, a mixture of polyesters made up of sesquiterpenoid acids esteried with hydroxy aliphatic
acids, is a well-known water-insoluble polymer. Owing to its high solubility in N-methyl pyrrolidone (NMP) it
is interesting to apply as a polymer matrix for solvent-exchanged in situ forming gel. The aim of this research
was to study the gel properties, drug release and antimicrobial activities against Staphylococcus aureus,
Escherichia coli, Candida albicans, Streptococcus mutans and Porphyromonas gingivalis of the in situ forming gels
prepared from bleached shellac dissolving in NMP to deliver the antimicrobial agents (doxycycline hyclate, metronidazole and benzyl peroxide) for periodontitis treatment. The solvent exchange between NMP and external
aqueous simulated gingival crevicular uid stimulated the dissolved bleached shellac transforming into the
opaque rigid gel. Increasing the amount of bleached shellac increased the viscosity of the system while still
exhibiting Newtonian ow and increased the work of syringeability whereas prolonging the drug release. The developed systems comprising 5% w/w antimicrobial agent showed antimicrobial activities against all test bacteria.
Thus the solvent exchange-induced in situ forming gels comprising of bleached shellac-antimicrobial drugs exhibited potential use for periodontitis treatment.
2015 Elsevier Ltd. All rights reserved.

1. Introduction
Injectable in situ forming gels are particularly attractive for the delivery of drugs into the periodontal pocket for periodontitis treatment because they are in sol form before administration into the body and
gradually alter to a gel form or solid-like depot [1]. The treatment of
periodontitis by these intra-pocket antibacterial delivery systems is interesting due to the prospects of maintaining effective high levels of
drug in the gingival crevicular uid for a prolonged period of time to
produce the desirable clinical benets [2]. Chitosan gels comprising
metronidazole demonstrated the effectiveness in the periodontitis
treatment [3]. Other various polymeric materials have been used as
polymer in situ forming systems such as chitosan/glucose 1-phosphate
[4], poly(ethylene carbonate) [5], hydroxyethylcellulose and polyvinylpyrrolidone [6], hydroxyethylcellulose and polycarbophil [7], gellan
gum and sodium alginate [8], and HPMC [9,10]. However, for the hydrophilic nature of the abovementioned polymers, the sustainable release
for the antibacterial agents was difcult. Recently, ethyl cellulose more
prolonged the release of antimicrobial drugs when the higher concentration of this polymer was added in the in situ forming gel [11].
Corresponding author.
E-mail addresses: thawatchaienator@gmail.com, tphaechamud011@yahoo.com
(T. Phaechamud).

http://dx.doi.org/10.1016/j.matdes.2015.09.138
0264-1275/ 2015 Elsevier Ltd. All rights reserved.

Shellac is classied as generally recognized as safe (GRAS) material

which is the resin consisting mainly of polyesters made up of
sesquiterpenoid acids esteried with hydroxy aliphatic acids [12] (Fig.
1(left)). A major sesquiterpene in the structure is jalaric acid along
with a smaller proportion of laccijalaric acid [13,14]. Shellac is nontoxic,
physiologically harmless and biodegradable resin thus it is allowed to be
used commercially as a wood coating, a binder and an encapsulate for
pharmaceuticals and food supplements, and in cosmetic products [15
17]. Bleached shellac (BS) is obtained by dissolving shellac in sodium
carbonate solution and treating with sodium hypochlorite. It is a wellknown water-insoluble polymer dissolved in an organic solvent or solvent mixture for producing water-insoluble lms [18,19]. Shellac is
widely used as a moisture barrier coating for tablets and pellets due to
its notably low water vapor and oxygen permeability [20,21]. Type of
salt formation of shellac can affect the disintegrating properties of the
coated dosage forms [22] Oleogel and oleogel-based emulsions have
been prepared using shellac as structurant [23]. Colon-targeting shellac
nanobers have been fabricated with coaxial electrospinning process
[24]. However the in situ forming gel prepared with bleached shellac
has not been reported previously.
The ideal solvent for in situ forming gel needs to posses suitable
properties in terms of water afnity, viscosity and ability to dissolve
the drug and polymer [1]. For solvent exchange-induced in situ forming
gel the polymer and drug are initially dissolved in a water-miscible

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295

Fig. 1. Chemical structures of shellac (left) and N-methyl-2-pyrrolidone (NMP) (right).

organic solvent with low toxicity, such as 2-pyrrolidone, N-methyl-2pyrrolidone (NMP) or dimethyl sulfoxide (DMSO). The exchange between solvent moving from the system to the outside and diffusion of
aqueous from surrounding environment into the system resulted in
precipitation or solidication of polymer matrix [25]. NMP (Fig. 1
(right)) is used as solvent in this study because it is thermally stable,
biocompatible and can be used as an attractive solubilizer in the pharmaceutical eld [26]. NMP at single oral doses did not lead to an increase either in micronucleated erythrocytes or in structural or
numerical chromosomal aberrations when bone marrow was after
treatment in the micronucleus test or karyotype analysis [27]. NMP
has already been used for in situ forming depots formulation due to
pharmaceutical precedence in approved parenteral products [1,28,29].
Periodontitis resulting from bacterial infection could be used the antibiotics for treatment [30]. Doxycycline hyclate (DH) is one of bacteriostatic antibiotics used for periodontal therapy which its mechanism
of action is the interference of a bacterial protein synthesis [31]. Atrigel
is a commercial injectable periodontal pocket delivery system containing 10% doxycycline hyclate using poly(DL-lactide) as polymer dissolved
in NMP [32]. Metronidazole (MT) is bactericidal against anaerobic bacteria. It has been proposed that MT is intracellularly activated by reduction and the toxic effect of reduced intermediates bind to DNA leading
to loss of helical structure, strand breakage and impairment of DNA
function and it also has been used in the eld of periodontal therapy
[33]. Benzoyl peroxide (BP) consists of two benzoyl peroxide groups
bridged by a peroxide link which it releases the free radical oxygen species capable of oxidizing bacterial proteins [34]. BP in Eudragit RS systems containing peppermint oil has been developed for periodontitis
treatment [35]. The chemical structures of these three antimicrobial
drugs are presented in Fig. 2.
The aim of this study was to prepare the novel in situ gel forming systems of antimicrobial agents for periodontitis treatment. The in situ gel
formulations containing BS, drugs (DH, MT and BP) and solvent
(NMP) were prepared and investigated for their physical properties
and biological action as appearance, pH, viscosity, rheology,
syringeability, gel formation, rate of water diffusion into the gels, degradation and antimicrobial activity against Staphylococcus aureus,
Escherichia coli, Candida albicans, Streptococcus mutans and
Porphyromonas gingivalis.

2. Materials and methods

2.1. Materials
Bleached shellac (BS) (Ake Shellac Co., Ltd., Lumpang, Thailand) was
used as received. DH (Batch No. 20071121, Huashu Pharmaceutical Corporation, Shijiazhuang, China), BP and MT (T.MAN Pharma Ltd., Part,
Bangkok, Thailand) were used as model drugs. Brain Heart Infusion
(BHI) (lot no. 0270845, Bacto, USA), Brain Heart Infusion Agar
(BHA) (lot no. 0298038, Bacto, USA), Mitis Salivarius Agar (MSA)
(lot no. 0118681, Difco, USA), Sabouraud Dextrose Agar (SDA) (lot
no. 7312647, Difco, USA), Sabouraud Dextrose Broth (SDB) (lot no.
6345690, Difco, USA), Tryptic Soy Agar (TSA) (lot no. 7341698,
Difco, USA) and Tryptic Soy Broth (TSB) (lot no. 8091999, Difco,
USA) were used as media for antimicrobial test. The antimicrobial susceptibility test disk containing ampicillin 10 g/disk (Becton Dickinson
& Company, USA) was used as positive control for antibacterial test
and clotrimazole (kindly supported from T.MAN Pharma Ltd., Part,
Bangkok, Thailand) of 10 g/cup was used as positive control for antifungal test. N-methyl-2-pyrrolidone (NMP) (lot no. A0251390, Fluka,
New Jersey, USA) were used as solvent for BS. Potassium dihydrogen orthophosphate (lot no. E23W60, Ajax Finechem, Australia) and sodium
hydroxide (lot no. AF 310204, Ajax Finechem, Australia) was used as
component in phosphate buffer pH 6.8. A dialysis tube (Spectra/Por
membrane MWCO: 60008000, lot no. 32644, Spectrum Laboratories,
Inc., CAL, USA) was used as received.
2.2. Preparation of the in situ forming gel systems
BS (15, 20, 25 and 30% w/w) was dissolved in NMP (Table 1) and
kept for 24 h then a clear solution was formed. The 5% w/w antimicrobial agents (DH, MT and BP) were incorporated into the prepared systems
and kept for 24 h to obtain the clear solutions.
2.3. Evaluation of gel properties
2.3.1. Gel appearance and pH measurement
The appearance of prepared formulations, color and homogeneity
were observed by visual observation. The pH value of each formulation

Fig. 2. Chemical structures of doxycycline hyclate (left), metronidazole (middle) and benzoyl peroxide (right).

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T. Phaechamud et al. / Materials and Design 89 (2016) 294303

Table 1
Composition formula of various gel systems containing different drugs (5% w/w).
Amount (% w/w)
Formula
BS-1
BS-2
BS-3
BS-4
BS-5
BS-6
BS-7
BS-8
BS-9
BS-10
BS-11
BS-12

BS

NMP

DH

MT

BP

5
10
15
20
5
10
15
20
5
10
15
20

90
85
80
75
90
85
80
75
90
85
80
75

5
5
5
5

5
5
5
5

5
5
5
5

was measured using a at surface probe of pH meter (Ultra Basic UB-10,

Denver Instrument, Bohemia, New York) (n = 3).
2.3.2. Viscosity and rheological behavior studies
The prepared systems was determined for viscosity by using a
Brookeld DV-III Ultra programmable rheometer (Brookeld Engineering Laboratories Inc., Middleboro, MA, USA) with spindles (CP-40)(n =
3). Viscosity parameters were measured at different shear rates with
15 s equilibration time at every shear rate (n = 3). The viscosity measurements were made at 25 C and 37 C which were the room and
physiological temperatures, respectively. The shear stress of samples
was measured at various shear rates at 25 C and 37 C. The temperature
was maintained within 0.1 C by a water bath (Buchi Heating Bath B490, New Hampshire, USA) connected to a sample cup of the rheometer.
The samples were equilibrated on the plate to reach the running temperature prior to each measurement. The ow parameters were characterized using the exponential formula [36]:
FN G

Log G N Log FLog

where F is shear stress, G is shear rate, N is an exponential constant and

is a viscosity coefcient.
2.3.3. Syringeability test
Practically, syringeability of the systems is an important factor to
consider for the ease of administration by injection which is the force required to expel the prepared product via a needle. Syringeability of each
sample was evaluated using a texture analyzer (TA.XT plus, Stable Micro
Systems, UK) in compression mode. The sample was lled into a 1 mL
syringe with an 18-gauge needle that was clamped with a stand. Typically, the 18-gauge needle was widely used in the dental eld. The
upper probe of the texture analyzer moved downwards at constant
speed (1.0 mm s 1) until it came in contact with the syringe barrel
base. A constant force of 0.1 N was applied to the base and the distance
required to expel the contents for a barrel length of 20 mm was measured at room temperature (n = 3). Force displacement proles were
performed, which the force at distance of 10 mm were selected for
Table 2
pH values of BS systems containing various drugs (5% w/w).
pH S.D. (n = 3)
BS (% w/w)

With drug (5% w/w)

Without drug

15%
20%
25%
30%

7.22 0.01
7.02 0.03
6.81 0.02
6.65 0.02

DH

MT

BP

4.16 0.05
4.09 0.01
4.13 0.01
3.93 0.04

7.69 0.04
7.34 0.02
7.06 0.02
6.84 0.01

7.83 0.01
7.70 0.03
7.46 0.01
7.05 0.04

Fig. 3. Flow curve of bleached shellac formula containing 5% w/w DH at (A) 25 C and
(B) 37 C. Open symbols represent the up-curve, and closed symbols represent the
down-curve.

analysis. The area under the resulting curve (AUC) was used to determine the work of expulsion.

2.3.4. In vitro gel formation

Gel formation was determined by injecting the 1 mL sample
into 5 mL phosphate buffer solution pH 6.8 (to simulate the gingival crevicular uid) [37] with the 18-gauge needle. The turbid gel or solid-like
formation was observed visually and recorded at various times (0, 1, 5
and 30 min).
Table 3
Flow parameters of BS (1530% w/w) formula containing different drugs at 25 C and 37 C
(n = 3).
25 C

37 C

Flow index (N)

(mean S.D.)

Consistency
index ()
(mean S.D.,
[D/cm2]N s)

Flow
index (N)
(mean S.D.)

Consistency
index ()
(mean S.D.,
[D/cm2]N s)

DH
15%
20%
25%
30%

0.99 0.01
1.02 0.01
1.00 0.02

17.80 5.91
31.77 2.19
181.80 13.99

1.03 0.02
1.02 0.01
1.04 0.01
0.99 0.01

4.98 0.74
13.03 0.15
55.13 2.14
258.30 24.42

MT
15%
20%
25%
30%

0.98 0.01
0.97 0.01
0.96 0.01

33.53 5.15
57.33 7.32
224.57 6.04

0.99 0.02
0.97 0.04
0.98 0.01
0.98 0.05

11.50 1.01
29.00 3.96
97.17 16.74
354.47 26.86

BP
15%
20%
25%
30%

0.99 0.01
0.98 0.02
1.01 0.03

29.57 0.98
58.00 15.00
160.03 10.79

1.01 0.04
1.03 0.03
1.01 0.03
0.99 0.02

9.38 2.89
17.17 1.54
52.97 9.73
181.67 19.99

BS
(% w/w)

Not determined.

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297

release proles were tted with different mathematical release equations. Least square tting the experimental dissolution data to the mathematical equations (power law, zero order, rst order and Higuchi's)
was carried out. The high value of coefcient of determination (r2) or
model selection criteria (msc) indicated a superiority of the release prole tting to mathematical equations [38].

Fig. 4. Syringeability of BS formula containing different drugs.

2.3.5. Rate of water diffusion into the gels

The determination of water diffusion rate into the prepared systems
was performed by measuring the distance of the change from sol into
the turbid gel phase in a transparent tube (diameter 6 mm) immersed
in phosphate buffer solution pH 6.8 (15 mL) in a test tube. The distance
of water front diffusion was observed at various times (4 and 24 h). The
rate of water diffusion into the systems was calculated as in the following formula:
distance of water front diffusion mm:
Rate of water diffusion into the gels
:
time min

3
2.3.6. In vitro drug release studies
In vitro drug release studies were evaluated using both dialysis
membrane method and membrane-less diffusion method as previously
described [11] which the determination of drug release was performed
using a UVvis spectrophotometer at 349, 320 and 275 nm for DH, MT
and BP, respectively. All of the experiments were triplicately done, and
the mean cumulative drug release S.D. were calculated. The data obtained from the in vitro release were analyzed by a nonlinear computer
programme, Scientist for Windows, version 2.1. The cumulative

2.3.7. In vitro degradation studies

Degradation of the prepared gels was determined by incubating in
phosphate buffer pH 6.8. The 3 g sample was injected into phosphate
buffer pH 6.8 (10 mL). Each sample was incubated in the shaking bath
at 37 C with 50 rpm. Fresh phosphate buffer solution was replaced
every week for 1 month. Then the sample was dried in a hot air oven
at 65 C for 72 h and kept in a desiccator. The percentage of weight
loss was carried out as in the following:

%weight loss

Initial weightFinal weight

 100:
Initial weight

2.3.8. Antimicrobial activity studies

Antimicrobial activities of the prepared in situ forming gels were evaluated for both the standard microbes (S. aureus ATCC 6538P, E. coli ATCC
25922 and C. albicans ATCC 17110) and anaerobic microbes (S. mutans
ATCC 27175 and Porphyromonas gingivalis ATCC 33277) using agar-cup
diffusion method. The actively growing broth culture of microbes was
prepared with the turbidity approximately 108 cells/mL. Then, the swab
spread onto the agar plate and dried. The sterilized cylinder cups were
carefully placed on the surface of the swabbed agar. The prepared gels
were lled into the cylinder cup (8 mm diameter and 10 mm height)
and incubated at 37 C for 2448 h. For the anaerobic bacteria the test
was conducted in an anaerobic incubator (Forma Anaerobic System,
Thermo Scientic, Ohio, USA). The antimicrobial activities were measured
as the diameter (mm) of inhibition zone (n = 3).

Fig. 5. In vitro gel formation of BS (1530% w/w) formula containing 5% w/w DH at various times (0, 1, 5 and 30 min).

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T. Phaechamud et al. / Materials and Design 89 (2016) 294303

Fig. 6. In vitro gel formation of BS (1530% w/w) formula containing 5% w/w MT at various times (0, 1, 5 and 30 min).

2.3.9. Statistical analysis

All experimental measurements were collected in triplicate. Values
were expressed as mean S.D. Statistical signicance of the measurements was examined using one-way analysis of variance (ANOVA)
followed by the least signicant difference (LSD) post-hoc test or Duncan. The signicance level was set at p b 0.05.

3. Results and discussion

3.1. Gel appearance and pH measurement
BS formula containing DH, MT and BP (5% w/w) were yellow, light
yellow and light yellow liquid, respectively. The pH values of BS formula

Fig. 7. In vitro gel formation of BS (1530% w/w) formula containing 5% w/w BP at various times (0, 1, 5 and 30 min).

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299

Table 4
Effect of polymer amounts in the formula containing different drugs on rate of water diffusion into gels (n = 3).
BS
(% w/w)

15%
20%
25%
30%

Rate of water diffusion into gels (mm/min) (mean S.D.)

With DH (5% w/w)

With MT (5% w/w)

With BP (5% w/w)

At 4 h

At 24 h

At 4 h

At 24 h

At 4 h

At 24 h

0.0049 0.0012
0.0056 0.0012
0.0049 0.0012
0.0049 0.0012

0.0029 0.0005
0.0032 0.0002
0.0025 0.0002
0.0025 0.0004

0.0042 0.0021
0.0035 0.0012
0.0042 0.0021
0.0028 0.0012

0.0032 0.0005
0.0032 0.0002
0.0030 0.0004
0.0030 0.0004

0.0049 0.0012
0.0028 0.0012
0.0042 0.0000
0.0028 0.0012

0.0028 0.0003
0.0025 0.0002
0.0025 0.0002
0.0025 0.0002

without and containing 5% w/w DH, MT and BP are shown in Table 2.

The pH of formula without drug was decreased as BS amount was increased because this polymer is a mixture of polyesters made up of
sesquiterpenoid acids esteried with hydroxy aliphatic acids and a
major sesquiterpene in the structure is jalaric acid along with a smaller
proportion of laccijalaric acid [13,14]. Moreover the pH value of NMP
was 11.742 0.184 thus the pH was decreased as BS amount was increased because NMP amount was gradually decreased. The pH value
of DH was between 2.0 and 3.0 [39], whereas the pH of a saturated aqueous MT solution was 5.8 [40]. The pH of DH-loaded BS systems was less
than the others owing to acidity of DH and lowering of pH was from the
higher amount of dissolved BS. Moreover, the pH of all systems tended
to decrease with the BS concentration dependence together with the
decrease of NMP amount.

3.2. Viscosity and rheological behavior

BS (1530% w/w) formula comprising DH exhibited Newtonian behavior (Fig. 3). All formula showed the Newtonian ow, indicating the
up curve did coincide with the down curve. Data of systems containing
MT and BP were not shown however they showed the same trend.
However the viscosity of DH-loaded systems was notably higher than
that of MT- and BP-loaded systems. This might be owing to the charge
interaction between positive charge of doxycycline molecule and carboxylic acid from BS resulting in the increment of viscosity of these systems. While the apparent viscosities of BS (30% w/w) formula
comprising DH, MT and BP could not be determined due to its high viscous texture at 25 C. The shear stress of all formulations at 37 C was
lower than that at 25 C which the curves of formulations moved to a
lower shear stress value indicating loose structure. The viscosity measurement was determined at 25 and 37 C to understand its viscosity
and ow behaviors during administration by injection at room temperature (25 C) and after injection into gingival crevicular uid of periodontal pocket of human body (37 C), respectively. Typically, the
viscosity of solution is decreased as the temperature is higher therefore
the viscosity of systems at 37 C was lower than that at 25 C. The viscosity of systems was increased when incorporated with drugs since drugpolymer interaction [41]. The shear stress of all drug-loaded formula
was higher than that of system without drug (data not shown). The
shear stress of all formula comprising 5% w/w drug was increased as
the shear rate and polymer amount were increased. The curves moved

to a higher shear stress value indicating compact structure of the gels.

The rheology and structure of entangled polymer systems could be altered by changing in the number of molecular entanglements [42].
The rheological behaviors of solutions comprising 5% w/w DH, MT and
BP were conrmed by the N value and viscosity coefcient () as
shown in Table 3. N value of all formula was close to 1, indicating Newtonian ow similar to BS solution without drug. In the case of the viscosity coefcient (), when the amount of BS in each formula was higher,
the value was also signicantly higher (p b 0.05). Temperature did
not inuence the ow types however the value at 25 C was signicantly less than that at 37 C (p b 0.05) due to the loose structure at
higher temperature.
3.3. Syringeability
The administration of the in situ forming system can be easily and
rapidly carried out, without pain, by using the proper needle and syringe [43]. The force required to expel the product through the needle
could be determined from the syringeability. The work of syringeability
of all formula signicantly increased (p b 0.05) as the amount of polymers was increased (Fig. 4). The increasing polymer content increased
the work required for expulsion, indicating lower syringeability as previously reported [44]. Notably, the apparent high injectability of DHloaded system was evident. This might be owing to the charge interaction between positive charge of doxycycline molecule and carboxylic
acid from BS promoting the increment of viscosity of systems as previously mentioned. Nevertheless, all prepared systems could expel
through the 18-gauge needle for administration by injection.
3.4. In vitro gel formation
This test was performed to observe the transformation of drug-loaded BS solution into gel or solid-like matrix owing to solvent exchange
mechanism. The in vitro gel formation of BS (1530% w/w) formula containing DH, MT and BP (5% w/w) in phosphate buffer pH 6.8 (simulated
gingival crevicular uid) are shown in Figs. 57, respectively. The 15%
w/w BS formula containing DH, MT and BP did not form the complete
gelation, while those of 20% w/w BS formula formed into solid-like
after injecting into PBS pH 6.8. The increased BS amount as 25 and
30% w/w, the formed gel was much more solid-like and opaque. The in
vitro injection experiments suggested that the polymer solution could

Fig. 8. Schematic of solvent exchange and formation of antimicrobial agent-loaded BS depot.

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T. Phaechamud et al. / Materials and Design 89 (2016) 294303

be easily injected into the periodontal pocket and formed in situ gel immediately which the higher polymer concentrations would quickly precipitate. Hence the lower amount of polymer did not result in the
gelation of the system, while increasing the polymer amount resulted
in the rapid precipitation as previously mentioned [45]. Moreover this
transformation also depended on the hydrophilicity of incorporated
drugs. The more hydrophilic drug as DH the more rapid transformation
was evident than that of hydrophobic drug such as MT and BP, respectively, because the higher wettability promoted the greater penetration
of water molecules to stimulate the solvent exchange and precipitation
of BS. The hydrophilic drug could act as a channeling agent, by rapidly
dissolving and easily diffusing outward, therefore allowing a decrease
in tortuosity and/or an increase in the matrix porosity for water penetration to expose the inner matrix [46]. Thus the transformation was apparently rapid for DH-loaded system.

Table 5
Comparison of degree of goodness-of-t from curve tting of the release proles of DH in
phosphate buffer pH 6.8 using dialysis membrane method (A) and membrane-less method (B) to different release models.

3.5. Rate of water diffusion into the gels

rate with prolongation of drug release [48]. Therefore, the aqueous diffusion and solvent exchange played the important role on drug release.

Rate of water diffusion into the gels of systems containing DH, MT

and BP tended to decrease when increasing polymer amount
(p b 0.05) (Table 4). The dense area with low water content could
lower the water diffusion coefcient [47]. The slower diffusion rate
was found at longer time of detection. When the solvent exchange initiated BS gradually transformed into solid-like depot and behaved like
a thicker barrier with time (Figs. 57) for the aqueous penetration
into inner system. The diffusion path was lengthened and the penetration of water was gradually slower with time. The schematic of solvent
exchange and formation of antimicrobial agent-loaded BS depot as previously described is presented in Fig. 8. Typically, the rapid solvent extraction was often followed by a fast drug release. On the other hand,
for the drugs of poor solubility, a rapid diffusion of DMSO would lead
to a fast solidication of the implant, resulting in a high drug retention

BS
(% w/w)

First order
r

Higuchi's
msc

Zero order
2

Power law

msc

msc

r2

msc

(A)
15%
20%
25%
30%

0.9552
0.9261
0.9179
0.9244

2.62
2.27
2.25
2.32

0.8798
0.8563
0.9277
0.9745

1.67
1.69
2.23
3.27

0.9245
0.8705
0.9235
0.9289

2.01
1.60
2.17
2.28

0.9953
0.9596
0.9727
0.9861

4.51
2.61
3.23
3.90

(B)
15%
20%
25%
30%

0.9971
0.9956
0.9989
0.9984

5.49
5.05
6.45
6.03

0.9932
0.9851
0.9809
0.9591

4.59
3.81
3.56
2.80

0.9698
0.9711
0.9899
0.9970

3.14
3.18
4.19
5.42

0.9970
0.9943
0.9985
0.9990

5.20
4.63
5.89
6.29

3.6. In vitro drug release studies

3.6.1. Dialysis membrane method
The drug release was tested in PBS pH 6.8 to simulate the environment of periodontitis. Drug release from BS systems was slower than
that from a DH solution using NMP as solvent without polymer addition
with about 90% drug release at 3 h whereas that of 5%, 10%, 15% and 20%
w/w BS systems were about 90%, 85%, 84% and 80% drug release at 6 h
(Fig. 9(A)). Drug release from the in situ gel systems decreased with increasing of the polymer amount. The physical entanglement between
polymers was able to control the drug diffusion. It has been reported
that the initial burst release was signicantly affected by polymer
phase inversion dynamics and the increase in polymer concentrations
could reduce the burst release [49]. Increasing HPMC concentration decreased the release rate of drug from HPMC matrices [50]. The release of
DH from three different polymer implants has been studied. The more
hydrophobic poly (dl-lactide co-caprolactone) (PLC) showed the
slowest release of drug, whereas the hydrophilic poly (DL-lactide-coglycolide) (PLG) led to low initial release of drug followed by a more
rapid release once the polymer becomes hydrated [51]. The solvent
type was one of critical factors inuencing the drug release [52]. NMP
promoted a rapid phase inversion associated with a high drug burst
due to the formation of a porous rubbery gel structure. When other solvents such as triacetin and ethyl benzoate, both weak solvents for PLGA
were used to signicantly slow gelation generated from the reduction of
burst release. Thus the diffusion of NMP provoked the burst drug release. Nevertheless, the high BS loading could minimize that effect and
the more sustainable drug release could be attained.
The membrane-less diffusion method was also conducted to allow
the simulated gingival crevicular uid to directly contact the gel surface
Table 6
Estimate parameter from curve tting of DH release in phosphate buffer pH 6.8 using dialysis membrane method (A) and membrane-less method (B) to power law expression.

Fig. 9. Effect of BS amount on release of DH using (A) dialysis method and (B) membraneless method (n = 3).

BS (% w/w)

k S.D.

n S.D.

Release mechanism

(A)
15%
20%
25%
30%

0.2821 0.0345
0.1131 0.0148
0.0700 0.0045
0.0507 0.0074

0.15 0.02
0.25 0.02
0.30 0.01
0.34 0.01

Fickian
Fickian
Fickian
Fickian

(B)
15%
20%
25%
30%

0.0062 0.0028
0.0043 0.0010
0.0019 0.0007
0.0007 0.0002

0.59 0.06
0.62 0.03
0.70 0.04
0.82 0.07

Anomalous
Anomalous
Anomalous
Anomalous

k = release rate; tl = lag time and n = diffusional exponent.

T. Phaechamud et al. / Materials and Design 89 (2016) 294303

Fig. 10. Percentage of weight loss of formula containing different drugs (n = 3).

whereas dialysis method that medium gradually diffused through dialysis membrane. The drug release from the formula decreased with an
increased polymer amount (Fig. 9(B)). Higher polymeric content in
the matrix decreased the release rate of drug because of the increased
tortuosity and decreased porosity [53]. The in situ forming gel comprising ethyl cellulose more prolonged the drug release when the higher
concentration of ethyl cellulose was incorporated [11]. The specic
properties of the network of polymer chains, e.g., the chain length,
their exibility and mobility, their water-uptake and swelling behavior,
extent of plasticization, or potential interactions between polymer and
drug would all potentially affect the diffusion rates in the polymer matrix and the drug release rate [54]. Moreover, the morphology change
during matrix formation could affect the drug release. By comparison
the drug release using membrane-less diffusion was slower than that
from dialysis tube method. The rapid transformation into BS matrix
owing to direct contact with dissolution medium promoted the hard
surrounding shell. This obtained dense surface of matrix resulted in
lowering of the water diffusion as previously described [11,47] and retardation of drug liberation. Moreover, BS was hydrophobic polymer
thus the drug hardly diffused passing this polymer and the sustainable
drug release was achieved.
3.6.2. Analysis of drug release data
The r2 and msc from curve tting to rst order, Higuchi's, zero order,
and power law equations after the release studies using dialysis

301

membrane and membrane-less methods are shown in Table 5(A) and

(B), respectively. DH release from all BS formula tted well with rst
order model. It has been reported that the drug release from hydrophobic polymer matrix was a rst order release [55]. The release exponent
values (n) from power law of drug release using dialysis membrane
method are shown in Table 6. The n value of the 510% w/w BS formula
signied that the drug release was by Fickian diffusion mechanism. The
drug release rate was decreased as a function of time due to a decrease
in the concentration gradient. The increased polymer amounts signicantly decreased the drug release rate (k) (p b 0.05) because the viscosity of the higher polymer amount formula was higher than that of lower
polymer amount formula.
DH released data from (515% w/w) BS using membrane-less
method were tted well with a rst order model (Table 6(B)). The n
value of all formula using membrane-less method indicated an anomalous (non-Fickian) diffusion controlled release (0.45 b n b 0.89) (Table
6(B)). Considering the drug release rate (k) parameter indicated that
the increased polymer amounts tended to decrease the drug release
rate (k) (p b 0.05) that was similar to the previous release study using
dialysis membrane method. The factors affecting release kinetic are liquid diffusion rate and polymeric chain relaxation rate. When the liquid
diffusion rate is slower than the relaxation rate of the polymeric chains,
the diffusion is Fickian, whereas when the relaxation process is very
slow compared with the diffusion, the case II transport occurs. When
liquid diffusion rate and polymer relaxation rate are of the same order
of magnitude, anomalous or non-Fickian diffusion is observed [56].
Solute transport from non-degradable polymeric systems is mainly
considered as diffusion driven. Non-degradable polymers can be
fabricated into reservoir- and matrix- type devices, which can be a
rate-controlling membrane [57]. For matrix-type devices, drug release
is more likely to be Fickian diffusion driven, which is associated with
concentration gradient, diffusion distance, and the degree of swelling
[58].
3.7. In vitro degradation studies
The percentage of degradation of BS with and without drug loading
signicantly decreased with increasing BS amounts (p b 0.05) (Fig. 10).
The degradation occurred by the diffusion of drug and NMP after exchange with medium. The degradation could be described by weight
loss or molecular weight loss [59]. Gingival crevice and periodontal

Fig. 11. Inhibition zone diameter of BS formula containing different drugs against S. aureus, E. coli, C. albicans, S. mutans and P. gingivalis (n = 3).

302

T. Phaechamud et al. / Materials and Design 89 (2016) 294303

pocket pH is more alkaline in gingivitis state [60]. Therefore the more

degradability should be provoked automatically for BS after injection.
The topography from SEM (data not shown) also conrmed this characteristic because the specimen after dissolution was collapsed and broken into debris. Thus BS demonstrated a high degradation and it has a
potential to diminish from the periodontal pocket after use. Shellac
showed an adequate cellular compatibility with human gingival broblasts and a signicant inuence on human on human dentin hydraulic
conductance indicating its safety and effectiveness as potential
desensitizing agent [61]. Moreover, shellac is nontoxic, physiologically
harmless and biodegradable resin [15]. Thus this designed depot system
has the potential use for delivering the antimicrobial drug with biodegradable characteristic.

3.8. Antimicrobial activity studies

The inhibition zone diameter of BS formulae against S. aureus, E. coli,
C. albicans, S. mutans and P. gingivalis are shown in Fig. 11. All systems
without drug loading exhibited the antimicrobial activities against test
microbes because of the antimicrobial activity of NMP [62]. NMP-loaded
ethylene oxidepropylene oxide block copolymer thermosensitive gel
exhibited antimicrobial activities against S. aureus, E. coli and C. albicans
with dose-dependent manner [62,63]. NMP is an acceptable pharmaceutical solvent [64] as solubilizing agent in parenteral and oral liquid
dosage forms [65]. The solubility parameter of NMP is similar to those
of ethanol and DMSO [66]. Therefore, NMP could solubilize the lipid in
cell membrane and promote the leakage of microbial cell membranes.
The inhibition zone of DH-loaded systems was signicantly larger
than that of the gel base (p b 0.05). In addition they showed the highest
activity signicantly against all tested microorganisms except C. albicans
because this organism was not susceptible with DH. MT-loaded systems
displayed a bactericidal activity against anaerobic bacteria both S.
mutans and P. gingivalis. Practically, MT has been used in the eld of
periodontal therapy [33] and solvent type could inuence the antimicrobial activity of MT-loaded in situ forming gel [67]. BP-loaded systems
displayed the antimicrobial activities against both C. albicans and P.
gingivalis from releasing the free radical oxygen species capable of oxidizing microorganism proteins [34]. BP-loaded in situ forming gel containing peppermint oil was applied for periodontitis treatment [35].
When BS amount was increased from 15% w/w to 30% w/w, the inhibition zone diameter against all microbes signicantly decreased
(p b 0.05) due to the higher compact matrix retarded the drug release.
The drug gradually released and prolonged regarding the increasing of
tortuosity and decreasing porosity owing to the higher polymeric content in the matrix [53].

4. Conclusion
DH, MT and BP loaded-BS systems comprising NMP as solvent were
formed into the in situ forming gel in simulated gingival crevicular uid
when the solvent exchange and polymer precipitation were occurred.
Gel formation capacity depended on the BS amount. The more sustainable drug release was achieved as increasing amount of BS. DH, MT and
BP loaded-BS systems effectively inhibited S. aureus, E. coli, S. mutans
and P. gingivalis therefore they exhibited the potential use as localized
delivery systems for periodontitis treatment.

Acknowledgments
This research work was grateful for the Research and Development
Institute, Silpakorn University (Grant No. SURDI 57/01/42). This research work was also facilitated by the Faculty of Pharmacy, Silpakorn
University, Thailand.

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