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ISSN: 1083-7450 (print), 1097-9867 (electronic)

Pharm Dev Technol, Early Online: 1–11

! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/10837450.2014.920361

RESEARCH ARTICLE

Optimization studies on development and evaluation of papain-based

in situ gelling system for chemomechanical caries removal
Ravi Shankar Tripathi and Kamla Pathak
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Department of Pharmaceutics, Rajiv Academy for Pharmacy, Mathura, Uttar Pradesh, India

Abstract Keywords
Context: Chemomechanical caries removal is a non-invasive technique that eliminates infected In situ periodontal gel, micro-tensile bond
dentine via a chemical agent. Papain, owing to its proteolytic nature causes disruption of strength, optimization, papain, preclinical
degraded collagen fibrils that helps easy removal of the caries and has both bacteriostatic and evaluation
bactericidal action.
Objective: The objective of the present work was to formulate and evaluate papain-based in situ History
gelling system for chemomechanical caries removal, based on the concept of pH-triggered
in situ gelation and evaluate its pharmaceutical and chemomechanical characteristics. Received 2 April 2014
Material and methodology: A 32 full factorial design was employed to formulate the in situ gels. Revised 21 April 2014
Carbopol 934 and HPMC K15M were designated as two independent variables, each utilized at Accepted 23 April 2014
three different levels and the dependent variables were gelling capacity, viscosity and % Published online 20 May 2014
cumulative drug permeated (CDP). The optimized formulation was assessed for ex vivo clinical
For personal use only.

efficacy by SEM, micro-tensile bond strength and antibacterial activity.

Results: Formulation F3 with % CDP of 10.13 ± 0.43% and optimum gelling and viscosity
characteristics was optimized. The efficacy of F3 was confirmed by enhanced micro-tensile
bond strength of 38.48 ± 4.14 Mpa compared to 29.42 ± 2.33 Mpa of control group and SEM.
Conclusion: An economically viable papain-based in situ gelling system with clinical potential
for caries removal with enhanced bonding ability was successfully developed.

Introduction vibration and noise5. Thus, an effective system for caries removal
should be identified that can differentiate between these layers
Dental caries is one of the most common endogenous infectious
and removes only outer necrotic layer for cavity preparation for
diseases afflicting mankind. Dental caries is an obstinate;
adhesive restoration material to stop the pathology of dental caries
dietomicrobial, multi-factorial, site-specific disease which
and restoration of structure of tooth6.
involves localized destruction of mineralized tissue due to acids
The preservation of tooth tissue and philosophy of minimal
produced by the bacterial glycolysis of dietary carbohydrates1.
invasive dentistry led to the usage of chemomechanical caries
The infection starts from the outer surface of the tooth and
removal (CMCR). This new method is patient friendly and acts by
through dentine progresses toward the pulp cavity ultimately
causing further degradation of the partially degraded collagen in
compromising the vitality of the tooth (Figure 1). Carious dentine
the infected dentine (Figure 2). It promotes delivery of atraumatic,
consists of two distinct zones the outer infected dentine that is
bactericidal and bacteriostatic activity, while removing the least
irreversibly denatured and non-remineralizable and the inner
amount of tooth structure7. The CMCR agents are chemical
affected dentine that is re-mineralizable and is reversibly
agents which causes further degradation of carious tissue and help
denatured2. Thus, the differentiation between the two layers is
in easy removal and tooth restoration.
clinically vital while treating the carious lesion so that the outer
Papain is an enzyme obtained from the leaves and fruits of
necrotic layer can be removed and the inner layer can be
Carica papaya. It is an endo-protein with bactericidal, bacterio-
preserved3.
static, anti-inflammatory and skin debridement activity8. Papain
Conventional methods of caries excavation are performed by
cleaves collagen molecules partially destroyed by the action of
using rotary and sharp-edged hand instruments (Figure 2)4. These
caries, and is able to digest dead cells, eliminating the fibrin coat
have various disadvantages, for example, removes healthy tooth
formed by the caries process resulting in easy removal of caries.
structure in addition to the decayed areas, mechanical preparation
It acts only on carious tissue which lacks the plasmatic protease
often induces pain and discomfort and there is generation of heat,
inhibitor alpha-1 antitrypsin, but its proteolytic action is inhibited
on healthy tissue, which contains this substance9. Papain is safe,
non-cytotoxic and biocompatible with oral tissues10.
Thus, the aim of the study was to develop pH sensitive in situ
Address for correspondence: Dr. Kamla Pathak, M. Pharm., Ph.D.,
Professor and Head, Department of Pharmaceutics, Rajiv Academy for
gelling system for localized targeted delivery of papain at the
Pharmacy, National Highway No. 2, Chattikara PO, Mathura, 281001, target site with enhanced retentivity. The proposed system will be
Uttar Pradesh, India. Fax: +91-565-2825050. E-mail: kamla_rap@ in solution form at low pH allowing easy administration and will
yahoo.co.in form a gel at physiological oral pH of 6.8 with sufficient adhesion
 2 R. S. Tripathi & K. Pathak
force leading to increased concentration at target site and less
washout. The objective was to formulate and evaluate the
pharmaceutical parameters of the in situ gels using 32 full
factorial design and to determine the clinical efficacy and
chemomechanical parameters of the optimized formulation.
A 32 randomized factorial design studies the simultaneous
influence of two formulation factors at three different levels
(1, 0 and 1) that are termed as independent variables on the
desired formulation characteristics (dependent factors). The
results of trials were evaluated using Design-Expert Software
(version 8.0.7.1; Stat-Ease, Inc., Minneapolis, MN) and analyzed
statistically.
Materials and methods
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Materials
Papain IP was a gift sample from Shri Ganesh Industrial
Enzymes, Burhanpur, Madhya Pradesh, India. Carbopol 934P
and HPMC K15M were procured from Qualigens Fine Chemicals,
Mumbai, Maharashtra, India. L-Cysteine hydrochloride and
ethylenediaminetetraacetic acid were purchased from SD Fine
Chemicals Ltd, Mumbai, Maharashtra, India. Procurement of
casein and trichloroacetic acid were carried out from Titan
For personal use only.
Figure 1. Cross-sectional structure of teeth and progression of caries.
Figure 2. Schematic representation of
the caries removal by excavation and
chemomechanical method.
Pharm Dev Technol, Early Online: 1–11
Biotech Ltd, Bhiwadi, Rajasthan, India. Methylparaben and
propylparaben were obtained from S. Merck (Mumbai, India)
Ltd. Dialysis membrane (pore size: 0.22 mm) was purchased from
Hi Media Laboratories Pvt Ltd, Mumbai, Maharashtra, India.
Remaining solvents and chemicals used were of analytical reagent
grade.
Preliminary studies
Kinetics
Kinetic study of papain was carried out in aqueous media at
25 ± 0.5  C to analyze the remaining amount of enzyme after
regular intervals. Two sample solutions were prepared, first
containing papain (0.5% w/v) with no added excipients and the
second having papain (0.5% w/v), an antioxidant (sodium bisulfite
0.1% w/v) and complexing agent (EDTA 0.1% w/v). The amount
of enzyme after regular time interval was assessed with the help
of UV-Visible Spectrophotometer (UV-1700 PharmaSpec;
Shimadzu Corporation, Kyoto, Japan) operated in kinetic mode
at 278 nm.
Equilibrium solubility
Equilibrium solubility studies were aimed to analyze the solubil-
ity of papain in double-distilled water, phosphate buffer pH 6.8,
phosphate buffer pH 7.4 and acetate buffer pH 4.5. Excess amount
of papain was added to 10 ml of media separately in each conical
flask to ensure saturation. The flasks were shaken for 72 h in a
shaking water bath (HICON Enterprises, New Delhi, India)
maintained at 25 ± 0.5  C. Samples were withdrawn, filtered
through 0.2 mm size filter paper and assayed spectrophotometric-
ally by UV-Visible Spectrophotometer at 278 nm after suitable
dilution.
Selection of medium and gelling polymer
The study was conducted to select a suitable medium and polymer
or polymer combination for the formulation of pH-dependent
in situ gelling systems. Aqueous dispersions of different concen-
trations of carbopol 934P, HPMC K15M and their combinations
were prepared as depicted in Table 1. The pH of the dispersions
was recorded by calibrated digital pH meter (HICON Enterprises,
New Delhi, India) at room temperature. Gelling capacity was
evaluated by the test-tube inversion method in which triethano-
lamine was added drop-wise to the prepared polymeric disper-
sions till pH 6.0 is reached and the tube was inverted to check the
formulation of gel.
 DOI: 10.3109/10837450.2014.920361 In situ gel of papain 3

Enzyme-excipient(s) compatibility Cysteine hydrochloride was added to the solution for enzyme
activation. Sodium metabisulfite (0.1% w/v) and EDTA (0.1%
The study was carried out to identify any incompatibility between
w/v) were added while stirring. Cysteine hydrochloride was added
the enzyme and different excipients. The enzyme was mixed with
further. HPMC K15M was added slowly to the solution with
the selected excipients like carbopol 934, HPMC K15M in ratio
continuous and low stirring to avoid air entrapment. Stirring was
1:5 and 6% moisture. The physical mixtures were kept in clear
continued till a viscous solution of uniform dispersivity is formed.
glass vials at 55  C for 2 weeks and vials were examined at regular
Finally, carbopol 934P was sprinkled over the solution and
intervals for discoloration, caking, liquefaction and odor. The
stirring continued for 12 h to get the formulation.
samples were analyzed by diffuse reflectance spectroscopy for
any chemical interaction. Samples of papain, carbopol 934,
Evaluation and characterization of in situ gelling system
HPMC K15M and their physical mixture were triturated separ-
ately with potassium bromide (IR grade) in the ratio of 1:9 and Clarity, pH and enzyme content
placed in the sample cup mounted in the instrument. The
The developed formulations were evaluated for clarity by visual
spectrographs were recorded in terms of transmittance mode (%T)
inspection against black and white background, and pH was
using FTIR Spectrophotometer (FTIR-8400; Shimadzu
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recorded by calibrated digital pH meter (HICON Enterprises,

Corporation, Kyoto, Japan) with DRS attachment (DRS-8000).
New Delhi, India) at room temperature. For enzyme content
Scanning range of 400–4000 cm1 with 1 cm1 resolution was
determination, a specified quantity of formulation was placed in
used.
10 ml of volumetric flask and the volume was made up to 10 ml
using phosphate buffer, pH 6.8. The volumetric flask was shaken
Experimental design
for 1 min using vortex shaker. The solution was filtered and the
A 32 randomized factorial design was used and two factors at filtrate was appropriately diluted with phosphate buffer, pH 6.8
three levels; were evaluated by experimental trials of nine and the concentration of papain in the filtrate was determined
possible combinations. The concentration of carbopol 934P (X1) spectrophotometrically at 278 nm.
and HPMC K15M (X2) were selected as independent variables as
depicted in (Table 2). Gelling characteristics, viscosity and % In vitro gelation
cumulative drug release (at 2 h) were selected as dependent
Gelling capacity/efficiency was determined by placing a drop of
variables. The resulting data was fitted into Design-Expert
the in situ gelling system in a vial containing 2 ml of phosphate
Software (version 8.0.7.1; Stat-Ease, Inc., Minneapolis, MN)
buffer saline pH 6.8 freshly prepared and equilibrated at
and analyzed statistically. 3D response plots were generated to
37 ± 2  C. The time of formation of gel and time taken for
estimate simultaneous influence of carbopol 934P and HPMC
For personal use only.

dissolution of the formed gel was recorded. The gel integrity, rate
K15M on dependent variables.
of formation of gel with respect to time were the factors used for
grading of the systems. The ‘‘’’ sign was assigned for no
Preparation of papain-based in situ gelling systems
gelation, ‘‘+’’ sign was assigned for gelation and immediate
The required amount of methyl hydroxybenzoate (0.20% w/v) and dissolution of gel, ‘‘++’’ sign was designated for gel having
propyl hydroxybenzoate (0.02% w/v) were added to 20 ml water. immediate gelation and stands for 1 h, ‘‘+++’’ sign was
The solution was heated upto 80  C to dissolve the preservatives designated for immediate stable gelation for hours.
and then cooled to room temperature. Accurately weighed papain
(10% w/v) was added to the solution and stirred well to dissolve. Viscosity
Viscosity before and after gelation at physiological pH 6.8 at
Table 1. Preliminary trials for selection of gelling polymers. 37 ± 0.5  C was determined using Brookfield viscometer (Model
DV-11 + PRO; Brookfield Engineering Laboratories, Inc.,
Carbopol HPMC Gelling Middleboro, MA) equipped with spindle T-bar (S-95). The
Trial code 934P (% w/v) K15M (% w/v) characteristics pH formulations were kept in 10 ml beaker and the spindle was
PF1 0.5 – No gelation 5.45 lowered perpendicularly taking care that the spindle did not touch
PF2 1.0 – No gelation 3.83 the bottom of the beaker. The spindle was rotated at 100 rpm to
PF3 – 0.5 No gelation 6.52 generate a torque of 450% and the readings were recorded after
PF4 0.5 0.5 Gelation 4.79 they became constant.

Table 2. 32 full factorial design for formulation of in situ gels of papain.

Sodium
Formulation Enzyme Carbopol HPMC metabisulfite EDTA Methylparaben (1:10)
code (% w/v) 934 (% w/v) X1 K15M (% w/v) X2 (% w/v) (% w/v) propylparaben (% w/v) Dependent variables
F1 10 0.5 (1) 0.2 (1) 0.1 0.1 0.2 Viscosity of sol
F2 10 1.0 (0) 0.2 (1) 0.1 0.1 0.2
F3 10 1.5 (+1) 0.2 (1) 0.1 0.1 0.2
F4 10 0.5 (1) 0.4 (0) 0.1 0.1 0.2
F5 10 1.0 (0) 0.4 (0) 0.1 0.1 0.2 Gelling characteristic
F6 10 1.5 (+) 0.4 (0) 0.1 0.1 0.2
F7 10 0.5 (1) 0.8 (+1) 0.1 0.1 0.2
F8 10 1.0 (0) 0.8 (+1) 0.1 0.1 0.2 In vitro permeation
F9 10 1.5 (+1) 0.8 (+1) 0.1 0.1 0.2
F10a 10 1.25 (+0.5) 0.3 (0.5) 0.1 0.1 0.2
a
Extra-design checkpoint formulation.
 4 R. S. Tripathi & K. Pathak Pharm Dev Technol, Early Online: 1–11

In vitro drug permeation on the base of the curved orthodontic brackets according to the
manufacturer’s instructions and photopolymerized for 20 s. The
In order to review the drug release behavior of in situ gelling
bracket was positioned with a seating force of 0.225–0.280 kgf4
systems in vitro, permeation study was carried out with Franz
with a Dontrix gauge (3M Unitek Dental Products, Monrovia,
diffusion cell. A cellulose nitrate membrane (0.22 mm, Hi Media
CA) and photopolymerized for 60 s (15 s from cervical, incisal,
LA 387-5MT; Hi Media Laboratories Pvt Ltd, Mumbai,
mesial and distal direction) to firmly attach the adhesive resin to
Maharashtra, India) was hydrated in phosphate buffer, pH 6.8
the dentine surface.
for 24 h before use. Cellophane membrane was tied to one end of
The hemisected specimens of group G2 were treated with the
the donor cell. One ml of formulation was accurately transferred
optimized formulation for 60 s. The gel was removed with the
to the cell. The donor cell was immersed in 10 ml of phosphate
help of a stainless steel small-spoon hand excavator until no
buffer, pH 6.8 in receiver compartment. The system was
residues of gel could be detected visually. The dentine surface was
maintained at 37 ± 0.5  C and magnetically stirred. Aliquot of
cleaned with wet cotton pellet and air dried with oil free
1 ml was withdrawn from the receiving cell at intervals of 0, 5, 10,
compressed air for 10 s. The surface was etched with 37% w/v
20, 30, 60 and 120 min and replenished with equal quantity of
phosphoric acid with a microbrush for 15 s, washed with distilled
fresh phosphate buffer, pH 6.8. The enzyme permeated through
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water and then air dried with oil-free compressed air for 20 s. Rest
the membrane was estimated by measuring the absorbance at
of the procedure is same as described for G1 and the brackets are
278 nm.
attached as in group G1.
Micro-tensile tensile bond strength
Shear bond strength estimation
Extracted human third molars with occlusal moderate caries were
All the hemisected carious teeth with attached bracket (both group
stored in saline solution (0.9% w/v) at 4  C and used within
G1 and G2) were stored in distilled water at room temperature
1 week. After being scaled of the residual tissue, move plaque and
(37  C) for 24 h. Following which they were subjected to
other pit and fissure debris were removed. The crowns were
thermocycling of 500 cycles at 5 and 55  C prior to the estimation
examined for signs of clinical caries using visual inspection at
of shear bond strength. The exposure to each bath was 20 s and the
2.5 magnification. Any teeth showing signs of extraction
dwelling time between the baths was 5 s. Following thermo-
damage or extensive cavitated lesions with pulpal involvement
cycling, the shear bond strength of all the samples was measured
were discarded from the study. Teeth exhibiting signs of marked
on Universal Testing Machine using a stainless steel shear probe
cavitation on the occlusal surface were included and these were
with a crosshead speed of 1 mm/min. The minimum load required
sectioned longitudinally through the carious lesion, mesiodistally.
to produce the bond failure was determined from the first load
Twenty carious hemisected teeth were assigned to two different
For personal use only.

groups G1 and G2. The teeth in group G1 was excavated using bur
on a dental high-speed handpiece with copious water spray while
the teeth in group G2 were excavated by chemomechanical
method. The teeth were washed with water for 1 min and air dried
gently. Each tooth was mounted in a cylindrical stainless steel
mould of the dimension of (2.5  2.5 cm) with the help of self-
cure acrylic resin (Dental Product of India, Mumbai, Maharashtra,
India). The teeth were inserted within the mould such that the
crown portion of the tooth was completely visible (Figure 3).
The delineated carious surface of hemisected teeth of control
group G1 was treated with spherical steel bur for caries removal.
After the complete removal of the caries, the surface was treated
with 37% w/v phosphoric acid with a microbrush for 15 s, washed
with distilled water and then air dried with oil-free compressed air
for 20 s. A single coat of adhesive resin (Transbond XT 3M/
Unitek, Monrovia, CA) was applied onto the etched surface and
drop on the load deflection plot. The shear bond strength values
were calculated in megapascals (MPa) using the following
equation:
Bond strength ¼ force=surface area of the base of the bracket
ð1Þ
Scanning electron microscopy investigation
Teeth exhibiting signs of marked cavitation on the occlusal
surface were selected and these were sectioned longitudinally
through the carious lesion, mesiodistally. Twenty carious hemi-
sected teeth were randomly divided into four groups (i) dentine
treatment with conventional bur system, (ii) dentine treatment
with papain gel, (iii) enamel treatment with conventional bur and
(iv) enamel treatment with formulation papain gel. Each group
contains five teeth. The groups (i) and (ii) were assigned for the
morphological evaluation of the dentine and the groups (iii) and
(iv) for the micro-morphology of the enamel surface after caries
removal and cavity preparation. The specimens were air dried;
gold sputter coated, and observed the ultrastructure of dentine
and enamel surfaces using scanning electron microscopy
(VP-SEM S-3400N; Hitachi High-Technologies, Europe).

Antimicrobial efficacy
Clinical procedure
Primary molars with a broad occlusal cavitated lesion showing
brown and softened dentine were selected and collected. The
caries removing gel was kept in refrigerator for storage and was
withdrawn for sampling just before 1 h of application.
(i) An initial sample of the carious material was removed
superficially from the lesion using a sharp-sterile spoon
excavator. It was immediately transferred into a sterile vial
Figure 3. Mesiodistally sectioned tooth mounted in acrylic resin mould. containing 1 ml saline.
 DOI: 10.3109/10837450.2014.920361
(ii) With the help of sterile spoon excavator the cries removing
gel was placed into the cavity so as to caries. After the
specified time of 5 min the carious dentine was gently
scraped with spoon excavator using very light pressure.
Fresh gel was reapplied and carious dentine scraped using
similar procedure, until the gel no longer turned opaque
after scraping. A second sample of dentine was then taken
from the cavity floor with another sterile spoon excavator
and transferred to another sterile vial containing 1 ml of
saline.
Microbial cultivation and evaluation
The dentine samples were processed in the microbiological
laboratory within 1 h of collection. Each sample was vortexed for
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30 s in order to dislodge the bacteria from the dentin. The
samples were then serially diluted to obtain 10–4 dilutions, and
0.1 ml of this dilution was inoculated onto two different agar
plates. The agar was used to determine the total viable count. The
plates were incubated anerobically at 37  C for 3 days. Then,
using a colony counter, the number of colonies was determined
per sample and expressed as colony-forming units (CFU)/ml. The
data obtained was tabulated and subjected to descriptive statistical
analysis. The Student’s t-test was used to find the significance of
the study.
Results and discussion
Preliminary studies
Kinetic study
For personal use only.
Papain is a proteolytic enzyme that has stability problems because
it contains cysteine group which is susceptible to oxidation in
aqueous solutions, and presence of bivalent ions enhances
degradation11. The study was carried out to assess the stability
of the enzyme in aqueous media and to study the affect of
stabilizers (antioxidant and or complexing agent) on the enzyme
solution stability. As shown by Figure 4, the stability of papain is
strongly influenced by the presence of stabilizers in the solutions.
Antioxidants are used in the concentration of 0.01–0.5% w/v for
aqueous solutions preferably in range of (0.1–0.5% w/v)12. The
lower limit of the range was selected and the results were positive.
The enzyme alone in aqueous solution underwent degradation
resulting in decrease in enzyme content but presence of sodium
metabisulfite (0.1% w/v) and EDTA (0.1% w/v) prevented the
oxidation of the enzyme and stabilized the papain solution.
Figure 4. Kinetics of papain in aqueous solution. (A) Enzyme solution
and (B) enzyme solution with stabilizers, sodium metabisulfite (0.1%
w/v) and EDTA (0.1% w/v); n ¼ 3.
In situ gel of papain 5
Thereafter, all studies were carried out using stabilizers in the
selected concentration.
Equilibrium solubility
Solubility study was carried out to assess the solubility of papain
in different media and select the suitable media in which papain is
soluble so that a uniform solution may be formed for formulation
of in situ gel systems. The study was carried out to investigate the
saturation solubility of papain to check the influence of pH
on solubility of papain. Three different media were selected
representing acidic, neutral and slightly basic conditions. The
equilibrium solubility of papain in double-distilled water and in
phosphate buffer pH 4.5, 6.8 and 7.4 was found to be 9.8 ± 0.146,
9.71 ± 0.123, 9.85 ± 0.114 and 9.82 ± 0.153 mg/ml, respectively.
The studies revealed that the solubility of the enzyme is pH
independent.
Selection of vehicle and gelling polymer
The pH of solutions plays a very vital role in oral in situ
pH-triggered gels. It influences the stability of the drug in the
solutions and also contributes to the safety and patient compliance
of the formulations13. The formulations should have a tolerable
pH, in the near neutral range (5–7.8). Carbopol 934 is widely used
polymer for the preparation of pH-dependent in situ gel systems.
It is an acrylic acid polymer, so it needs to be dissolved in a weak
acidic solution to prepare a free flowing liquid under non-
physiological conditions14. The dispersions containing 0.5% w/v
and 1.0% w/v carbopol 934 were not able to produce competent in
situ gelling system at pH 6.0 as detailed in Table 1. The pH 6.0
was taken instead of oral physiological pH of 6.8 as it is
considered that the pH of the microenvironment of the carious
tissue decreases due to acid produced by the oral microflora and
further to reduce the chances of non-gelation at pH 6.8. Increase
in concentration of carbopol 934 above a certain level is not
recommended as higher concentration of carbopol decreases the
pH of the dispersion below the tolerable limit of oral cavity. The
low pH can adversely affect the stability of the papain enzyme
having maximum activity in the pH range of 5.5–7.5 and also can
cause irritation and insensitivity to the oral cavity leading to non-
compliance. Hence a viscosity enhancing agent, HPMC was used
to enhance the gelling capability of carbopol 934 at oral
physiological pH 6.8 and also the resulting pH of the solutions
is expected to be in tolerable limit of oral cavity. Dispersions
containing 0.5% w/v of both carbopol 934 and HPMC possessed
good gelation capabilities. This observation can be attributed to
the formation of cross-links between the two polymers: the water
molecules completely hydrate the polymers and may act as a
cross-linking agent to form hydrogen bonds, which may lead to
the formation of 3D network and a strong gel structure can be
formed15.
Enzyme excipient incompatibility
No signs of discoloration, caking and liquefaction were found in
any of the samples or the physical mixture showing the absence of
physical incompatibility between the enzyme and excipients.
Papain (Figure 5A) showed one predominant band at 3500–
3200 cm1, having a peak at 3325 cm1 due to the N–H stretch of
a secondary N-substituted amide, C¼O stretch of a carboxyl
group at 1650 cm1, p-substituted aromatic out of plane C–H
deformation of an aromatic residue of tryptophan or tyrosine at
858 cm1 and strong peaks between 1150–1050 and 760–
570 cm1, having a peak at 1145 and 750 cm1 due to C–S
stretch of sulfides and disulfides16. Carbopol 934 (Figure 5B)
showed its characteristics peak in 3000–2950 cm1 range at
 6 R. S. Tripathi & K. Pathak Pharm Dev Technol, Early Online: 1–11

2987.53 cm1, corresponding to OH stretching vibration and
hydrogen bonding. The prominent peak between 1750 and
1700 cm1 at 1748 cm1 was attributed to carbonyl C¼O
stretching band. The band at 1250–1200 cm1 represented the
peaks due to acrylates. The prominent peak at 1160 cm1
corresponded to stretching vibration of ethereal cross-linking17.
HPMC K15M (Figure 5C) showed O–H vibration stretching peak
in range of 3500–3200 cm1, due to hydroxylpropyl group, the
peak between 1650 and 1600 cm1 at 1641.31 cm1 due to
stretching vibration of C–O of six membered cyclic rings. The
peak at 1000–950 cm1 is due to the presence of pyranose18.
In DRS spectra of physical mixture (Figure 5D), characteristic
peaks of papain and proposed formulation excipients were
Pharmaceutical Development and Technology Downloaded from informahealthcare.com by Miss Sandy Dalgleish on 11/10/14
retained with insignificant shift in peak signals, while the peak
at 3325 cm1 was broadened that may be probably due to
superimposed H–NH stretching of papain and O–H stretching
carbopol 934 and HPMC. The study confirmed absence of
chemical interaction between papain and formulation excipients
and were utilized for the formulation of pH sensitive in situ gel
of papain.
Formulation and evaluation
In situ gelling systems of papain were formulated using different
concentrations of carbopol 934 P and HPMC K15M, preserva-
tives, antioxidants and chelating agents and evaluated for various
parameters discussed below.

Appearance, pH and enzyme content

All the formulations appeared translucent with faint odor of raw
materials. Next, the pH that plays a pivotal role in oral
pH-triggered gelling system, affecting stability of the enzyme
and also influences the safety and patient compliance of the
product was assessed. The pH of all in situ gelling systems was
found to be in range of 5.02 ± 0.015 (F9) to 6.01 ± 0.083 (F1) at
25  C suggesting non-sensitizing and non-irritating attributes of
the developed formulations toward oral cavity. The enzyme
content of all the formulations was in range 92.35 ± 0.67 to
97.52 ± 0.61% w/v indicating uniform distribution of drug in the
formulations owing to single-step preparation of the system and
the results are shown in Table 3.

Viscosity
For personal use only.

The viscosity of formulations was determined before gelation at

Figure 5. Diffuse reflectance spectrograph of (A) papain, (B) carbopol
934, (C) HPMC K15M and (D) physical mixture of all components of
gelling systems.
simulated non-physiological condition and after gelation at
simulated physiological condition of pH 6.8 at 37 ± 0.5  C.
Maximum viscosity of 2380 cP was documented for formulation
F9 while the minimum viscosity of 140 cP was recorded for F1
before gelation at 25  C (Table 3). The viscosity increased
radically on reaching the physiological pH 6.8 at 37  C to 7050
and 460 cP for F9 and F1, respectively. As the pH of the system
increased to pH 6.8 there was a sudden increase in the viscosity
approximately to almost 300% suggesting significant sol–gel
phase transition between these systems before and after gelation.
The observed phase transitions for the in situ gelling systems can
be attributed to the ionization of carbopol polymer at physio-
logical pH of 6.8. At pH 6.8, mutual repulsion of ionized carboxyl
groups may produce more stretched carbopol backbone and those
carboxyl groups may also form stable hydrogen bonds with water
molecules through hydrophilic interactions19. While the hydro-
phobic nature of carbopol backbone that forms hydrophobic inter

Table 3. Evaluation of papain-based in situ gel systems, n ¼ 3.

Viscosity Viscosity Gelling

Code pH ± SD Enzyme content (before gelation) (cP) (after gelation) (cP) capacity
F1 6.01 ± 0.083 92.35 ± 0.67 240 460 
F2 5.47 ± 0.07 95.63 ± 0.51 420 890 +
F3 5.11 ± 0.032 96.06 ± 0.80 1080 2450 +++
F4 5.77 ± 0.020 96.02 ± 0.59 460 750 ++
F5 5.26 ± 0.051 97.52 ± 0.63 720 1460 ++
F6 5.04 ± 0.037 96.45 ± 0.73 1600 3480 +++
F7 5.42 ± 0.05 93.23 ± 0.38 1560 3800 +++
F8 5.19 ± 0.021 94.89 ± 0.52 1890 4790 +++
F9 5.01 ± 0.023 97.34 ± 0.61 2380 7050 +++
F10a 5.43 ± 0. 061 97.08 ± 0.43 950 2660 +++
a
Extra-design checkpoint formulation. ‘‘’’ sign indicates no gelation; ‘‘+’’ sign indicates that gelation occurs rapidly but dissolves
immediately; ‘‘++’’ sign indicates that immediate gelation occurs and stands for an hour; ‘‘+++’’ indicates that gelation occur
immediately and remained for extended period of time.
 DOI: 10.3109/10837450.2014.920361
chain aggregation, results in cross-linking forming more viscous
gel at physiological pH 6.820. The data indicated that viscosity of
in situ gelling systems was directly proportional to the concen-
tration of the polymers. As the concentration of carbopol 934 and
HPMC K15M were increased viscosity increased, this is in
correlation with literature reports21. The formulations should have
optimum viscosity in order to prevent drainage of the formulation
after gelation but boost in viscosity results in inconvenience
during delivery22. Thus the formulation F7, F8 and F9 were
rejected and rest of the formulations having intermediate viscosity
were selected for further evaluation due to easy flow ability and
subsequent ease of administration.
Gelling capacity
Pharmaceutical Development and Technology Downloaded from informahealthcare.com by Miss Sandy Dalgleish on 11/10/14
The two main pre-requisites of an in situ gelling system are the
viscosity and gelling capacity. Optimum viscosity is required for
easy administration and applicability and further on contact with
the physiological fluid of the desired pH the formulation should
undergo rapid transition from sol form to gel with enhanced
viscosity and sufficient adhesion properties. Formulation F8 and
F9 showed highest gelling capacity and formulations F3, F6, and
F7 indicated good gelling capacity while the formulations F1, F2
and F4 did not exhibit desirable gelling characteristics (Table 3).
Aqueous dispersions of carbopol transforms into stiff gel when its
pH is raised contributing its role in formation of pH-triggered
in situ gelling system23 of high viscosity and bioadhesive
property. The formulations having low concentrations of carbopol
were not able to show good gelling capabilities (F1) but as the
concentration of carbopol was increased gelling capacity of
For personal use only.
the formulations increased (F3). On inclusion of HPMC K15M in
the formulation, good gelling characteristics were obtained at
lower carbopol concentration (F5). In spite of this potential
ability, the concentration of carbopol required for forming stiff
gels results in solutions of very low pH which are not easily
neutralized by the buffering action of physiological fluid so
inclusion of viscosity enhancing agents/polymers such as HPMC
is recommended. This also has contributing role toward the
bioadhesion properties of the gelling system24.
In vitro drug permeation
The permeation profile (Figure 6) provides an insight into the
efficiency of the in situ gelling systems to release the enzyme and
the effect of carbopol 934 and HPMC on the behavior of the
Figure 6. In vitro permeation profiles of in situ gels of papain (F1–F9) in
phosphate buffer, pH 6.8 for 2 h at 37 ± 0.5  C using Franz diffusion cell;
n ¼ 3.
In situ gel of papain 7
system. The cellophane membrane (cellulose nitrate membrane
pore size: 0.22 mm) was used to hold the formulation and separate
the donor and receiver compartment only. Formulations F1
containing carbopol 934 (lower level) and HPMC K15M (lower
level) showed maximum enzyme permeation of 21.26% after 2 h,
while the formulation F3 containing carbopol 934 (higher level)
and HPMC K15M (lower level) permeated 10.18% of papain.
Formulation F9 containing both carbopol 934 and HPMC K15M
at higher level showed minimum permeation of only 2.03% after
2 h. The release behavior dictates that the cumulative amount of
enzyme permeated is dependent on the concentration of the
polymers. As the concentration of carbopol 934 and HPMC was
increased the viscosity of the formulation increased offering
enhanced diffusional resistance for the active and delaying the
release from the system25. Normally, the matrix systems display
an initial fast release because of the presence of drug at the
surface, but in this case the enzyme was first dissolved in the
media and then carbopol was added slowly which probably
resulted in complete entrapment of the enzyme in the matrix
structure of the gel leading to slow release of enzyme. Moreover,
at higher polymer contents the active enzyme is trapped in smaller
polymer cells and it is characterized by the close proximity of
polymer molecules to the enzyme. The density of chain structure
also limits the movement of the active26.
Selection of the optimized formulation
The optimized formulation was selected on the basis of gelling
capacity, viscosity and % cumulative drug permeated (CDP).
Based on optimum gelling capacity, formulations F3, F5, F6, F7,
F8 and F9 were screened for viscosity characteristics. Of these, F3
with viscosity values of 1080 cP at non-physiological pH was
screened out based on the consideration that viscosity in the range
of 150–1000 cP at storage conditions affords good flowability.
At physiological pH, the viscosity increased to 2600 cP confirm-
ing strong gelation. Furthermore, the % CDP of F3 was maximum
at 10.18%. Hence, the on the basis of good flowability, optimum
viscosity for easy administration, good gelation capability and
appropriate % in vitro permeation profile formulation (F3) from
within the experimental design was selected for further studies.
Statistical analysis
The response data was statistically analyzed with Design-Expert
Software (version 8.0.7.1; Stat-Ease, Inc., Minneapolis, MN).
A statistical model with individual and interactive terms in the
form of second order polynomial equation was used to evaluate
the response variables:
Y ¼ C0 þ C1 X1 þ C2 X2 þ C2 X22 þ C1 X21 þ C1 C2 X1 X2 ð2Þ
where X1 and X2 are independent variables and Y is the
dependent variable. C0 is the arithmetic mean response of nine
runs and C1, C2, C12 is estimated coefficients of corresponding
factors. The terms including X1 and X2 represent the effect of
single factor on the response variable on going from lower level
toward higher, while the interactive terms (X1X2) show the
combined effect of the two independent variables on the response
parameter. The mathematical equation generated for the quanti-
tative response parameters are expressed as:
% CDPðY1 Þ ¼ þ11:04  4:23X1  0:58X21  5:54X2  2:04X22
ð3Þ
ViscosityðY2 Þ ¼ 2788:89 þ 1118:8X1 þ 408:89X21
ð4Þ
þ 1522:2X2 þ 902:22X22 :
 8 R. S. Tripathi & K. Pathak
In Equation (3) negative sign of coefficient of X1 and X2
indicates inhibitory effect on the response, and the large
magnitude of coefficients showed that both the factors pronoun-
cedly reduces the enzyme release whereas in Equation (4) the
positive sign indicates that carbopol and HPMC K15M had
positive effects. The relationship between variables was further
elucidated using response surface plots. 3D bar surface chart
drawn for graphical optimization of papain-based in situ gel
systems clearly shows the effect of independent variables
carbopol and HPMC K15M level on the response variables.
It was observed that on increasing the levels of carbopol (X1) and
HPMC K15M (X2) simultaneously, the viscosity increased in a
pronounced manner (Figure 7A). While simultaneous increment
of carbopol and HPMC K15M has profound inhibitory effect on
Pharmaceutical Development and Technology Downloaded from informahealthcare.com by Miss Sandy Dalgleish on 11/10/14
the enzyme release from the formulations (Figure 7B).
Validation of experimental design
The polynomial equations were utilized for validation of the
experimental design for which an extra-design checkpoint
formulation (F10) was prepared and evaluated. Validation was
done by calculating % error (Table 4). The predicted value for the
two dependent variables % CDP (Y1) and viscosity (Y2) was
calculated from reduced polynomial equation and was compared
with experimental value. The difference between the two values
was calculated as % error. The low magnitude of % error proves
the validity of generated model in predicting response.
The linearity plots for predicted and observed response value
were also drawn for both dependent variables. The plot
for relationship between predicted and experimental viscosity
For personal use only.
(Figure 8A) and between predicted and experimental % CDP
(Figure 8B) were linear as described by significant r2 values
conforming the close concordance of the two values. This proves
the prognostic ability of the software to predict the values and
give optimized results.
Micro-tensile bond strength
CMCR approach involves the utilization of chemical agents to
remove the carious tissue leaving sound dentine for restoration by
filling materials to stop further progression of the process. After
removal of carious tissue, etching with 37% w/v phosphoric acid
is recommended to prepare the cavity for restoration creating
small pores and opening the dentinal tubules for resins. Adhesive
resins are utilized for the adhering the restorative material/filler
material to the dentinal surface of the cavity prepared for
restoration. The barrier layer between adhesive resin and dentinal
surface is the smear layer. The smear layer, which is an
Figure 7. 3D response plots showing simultaneous influence of independent variables on (A) viscosity and (B) % CDP of in situ gelling system of
papain within experimental design.
Pharm Dev Technol, Early Online: 1–11
amorphous layer of organic and inorganic debris, adheres firmly
to the dentinal surface and prevents resin from adhering to dentine
by occlusion of the dentinal tubules27.
Hence, removal of smear layer by chemical means is desirable
before mechanical procedure is ensued. Removal of smear layer is
assured by the measurement of micro-tensile bond strength.
Micro-tensile bond strength dictates the bonding ability of the
sound dentine surface after caries removal to the restorative
material/fillers so that efficient restoration can be developed and
for prolonged time. The micro-tensile bond strength for group
treated with mechanical bur and that treated with F3 formulation
were found to be 29.42 ± 2.33 and 38.48 ± 4.14 Mpa. Statistical
analysis of the values of micro-tensile bond strength shows
significant difference (p50.05). Micro-tensile bond strength of
the cavity after papain gel application was found to be much
higher than mechanical bur application. The enhanced bond
strength of group G2 may be attributed to removal of smear layer
more efficiently resulting in greater ability of the phosphoric acid
to reach the dentinal surface and penetrating deep to create pores
and partial dissolution of the surrounding peritubular dentine and
also leading to opening of tubules28. The adhesive resin molecules
micromechanically interlock with exposed dentine collagen fibre
network and resin tags into the opened dentine tubules. The resin
completely infiltrates the demineralized dentine, which creates a
hybrid or resin-reinforced dentine layer in the superficial
demineralized inter-tubular dentine matrix within the dentine
tubule29.
Morphological observation
The morphological characteristics of dentine surface have a
pronounced effect on the quality of adhesive bonding and
accordingly the longevity of the adhesive restorations. The
smear layer is an amorphous layer comprising of organic and
inorganic debris that occludes the tubules30. The layer has high
adhesive property for dentine surface preventing the adhesion of
resin to dentine after cavity preparation. Therefore, in order to
obtain an adequate bond to dentine, the smear layer must be
initially removed or treated prior to the placement of the
restoration conventionally by acid etching31.
Table 4. Evaluation of extra-design checkpoint formulation.
Evaluation parameters Predicted value Observed value (n ¼ 3)
% CDP 11.04 10.89 ± 0.23
Viscosity 2788 2667 ± 10.13
 DOI: 10.3109/10837450.2014.920361 In situ gel of papain 9
Pharmaceutical Development and Technology Downloaded from informahealthcare.com by Miss Sandy Dalgleish on 11/10/14

Figure 8. Predicted versus actual values graph for all the dependent variables, (A) viscosity and (B) % CDP to validate the design.
For personal use only.

Figure 9. SEM characterization after caries removal. (A) Enamel treated with phosphoric acid, (B) enamel treated with F3 formulation, (C) dentine
treated with phosphoric acid and (D) dentine treated with F3 formulation.

There are four types of pattern that explain the bonding
behavior of enamel tissue to the resin or adhesive system. Type I,
II, III and IV. In the first, called type I pattern, generalized
roughening of the enamel surface, but with distinct pattern
showing hollowing of prism centres with relatively intact
peripheral regions. In the second or type 2 pattern, prism
peripheries are removed or heavily damaged and the prism cores
project toward the enamel surface. Type III patterns represents the
region which did not conform to prism morphology. Type IV is
the half-hazard pattern comprising regions of both type I and type
II patterns32.
The micro-morphology of enamel surface showed type IV
pattern for treatment with conventional bur (Figure 9A) and type
II pattern was observed after treatment with formulation F3
followed by phosphoric acid (Figure 9B). Type I and type II
patterns have better bonding abilities to the adhesive material due
 10 R. S. Tripathi & K. Pathak
Table 5. Antimicrobial efficacy of papain-based in situ gel F3, n ¼ 3.
Pre-assessment of Post assessment of Reduction in
bacterial count bacterial count bacterial
Formulation (CFU/ml  104) (CFU/ml  104) count (%)
Placebo gel 18.3 ± 2.14 17.9 ± 2.34 2.1
F3 18.34 ± 2.51 03.76 ± 1.52 79.48
to infiltration of the resin tag into the hollow prism structure or
the peripheral linings having greater contact and bonding force
While type III and IV have weak bonding force for the restoration
materials33. SEM revealed the fact that mechanical bur method
was not able to remove the smear layer completely and the dentine
Pharmaceutical Development and Technology Downloaded from informahealthcare.com by Miss Sandy Dalgleish on 11/10/14
tubules were occluded by the layer (Figure 9C). Formulation F3
was able to remove the smear layer to a sufficient extent covering
the dentinal surface resulting in opening of dentinal tubules
clearly seen as small pores in (Figure 9D). SEM images also
explained the results obtained from micro-tensile bond strength
evaluation. The smear layer was removed to great extent after
application of papain-based gel system allowing more resin to
infiltrate into the dentinal tubules. The etching gel might have
penetrated deeper in the absence of (or presence of a very thin)
smear layer, within the same period of time which the acid had to
overcome the obstacle of smear before it could reach and interact
as deep with the underlying dentine34. The process resulted in
higher bond strengths of the resin and the dentinal surface as
found in micro-tensile bond strength evaluation. The principal
mechanism involved in the enhanced bonding ability of the resin
For personal use only.
to dentine surface is the combined effect of micromechanical
interlocking of resin molecules into an exposed dentine collagen
network and the tags of resin deep into the dentinal tubules35. The
results of the micro-tensile bond strength evaluation, morpho-
logical characteristics of dentine surface and micro-morphology
of enamel confirmed that papain-based gel can produce dentine
with greater bond strength.
Antimicrobial efficacy
The antimicrobial efficacy of papain-based in situ gel (F3) for
caries removal as an agent for chemomechanical approach
was evaluated (Table 5). A significant reduction of 79.48%
in the total viable count was observed in case of formulation
F3 as compared to the placebo gel. The result demonstrated
that papain has significant antibacterial activity against oral
cariogenic flora.
Conclusion
Papain was successfully formulated as pH-sensitive in situ gelling
system using carbopol 934 as pH sensitive polymer and HPMC
K15M as viscosity enhancing agent. The best formulation (F3)
possessed good flow ability at the formulated pH and
underwent rapid gelation when the pH of the formulation is
raised to physiological pH of 6.8. The system possessed easy
administration to the carious cavity and had high adhesivity
on coming in contact with the dentine surface leading to increased
retention and therapeutic efficacy of the system. F3 was able to
remove the smear layer from the dentine surface leading to
enhanced bonding ability of the dentine surface to the filling/
restorative material characterized by the high micro-tensile
strength and SEM. So, it can be concluded that removal of
carious tissue with a caries removing gel containing a natural
agent papain, proved to be efficient, easy to perform and
comfortable for the patient has a great potential for future
perspective.
Pharm Dev Technol, Early Online: 1–11
Declaration of interest
The authors report no conflicts of interest. The authors alone are
responsible for the content and writing of this article.
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