The Impact of Calcium Carbonate as Pore Forming Agent

and Drug Entrapment Method towards Drug Dissolution

Mechanism of Amoxicillin Trihydrate Encapsulated by
Chitosan-Methyl Cellulose Semi-IPN Hydrogel for Floating
Drug Delivery System
Fauzi Dewantara1, a) and Emil Budianto1, b)
1
Polymer Research Group, Department of Chemistry, Faculty of Mathematics and Natural Science, Universitas
Indonesia. Jalan Prof. Dr. Sudjono D Pusponegoro, Kampus UI Depok 16424, Depok
a)
first author: fauzidewa94@gmail.com
b)
second author: emilb@ui.ac.id

Abstract. Chitosan-methyl cellulose semi-IPN hydrogel is used as floating drug delivery system, and calcium carbonate also added
as pore forming agent. The hydrogel network arranged by not only using biopolymer chitosan and methyl cellulose, but also the
crosslink agent that is glutaraldehyde. Amoxicillin trihydrate entrapped into the polymer network with two different method, in
situ loading and post loading. Furthermore both method has been tested for drug entrapment efficiency along with drug dissolution
test, and the result for drug entrapment efficiency is in situ loading method has highest value of 100%, compared to post loading
method which has value only 71%. Moreover, at the final time of drug dissolution test shows in situ loading method has value of
96% for total accumulative of drug dissolution, meanwhile post loading method has 72%. The value of drug dissolution test from
both method is used for analyzing drug dissolution mechanism of amoxicillin trihydrate from hydrogel network with four
mathematical drug mechanism models as parameter. The polymer network encounter destructive degradation causes by acid
solution which used as dissolution medium, and the level of degradation is observed with optical microscope. However the result
shows that degradation of the polymer network doesn’t affect drug dissolution mechanism directly. Although the pore forming
agent causes the pore inside the hydrogel network create interconnection and it was quite influential to drug dissolution mechanism.
Interconnected pore is observed with Scanning Electron Microscope (SEM) and shows that the amount and area of interconnected
pore inside the hydrogel network is increasing as drug dissolution goes on.
Keywords: Amoxicillin Trihydrate, Drug Dissolution Method, Hydrogel, Pore Forming Agent.

INTRODUCTION
Bacteria is a microorganism that have many important role in our life and in our ecosystem, however there is some
bacteria has negative impact to humans life, one of them is Helicobacter Pylori which can cause an inflammation in
Gastro Intestinal Track (GIT). To neutralize the impact of the bacteria, high dose of antibiotic is used such as
Amoxicillin Trihydrate 1. High frequency consumption of the antibiotic causes drug concentration inside the body
become unstable and make the therapeutic effect is insufficient 2. To overcome the obstacles, controlled oral release
dosage form is used, with hydrogel network as a Floating Drug Delivery System (FDDS) 3.
FDDS can extend absorption time in the GIT effectively4. With the floating drug delivery system, a matrix
containing the drug will be floated and it will prolong the retention time in the stomach so that the drug can be released
completely with a controlled rate5. FDDS has been developed using a pore-forming agent and CO2 gasses could help
the system to float in the gastric fluid. Matrix is widely used in FDDS among others are hydrogels6. The hydrogel is
a smart material and is widely used as an application for the drug delivery. Hydrogels have been defined as two- or
multicomponent systems consisting of a three-dimensional network of polymer chains and water that fills the space
between macromolecules7.
 In this study, the monomer used as hydrogel network are chitosan and methyl cellulose which both have a non-
toxic properties for human body and insoluble with water. The hydrogel is made with semi-IPN method, using calcium
carbonate as Pore Forming Agent (PFA) and glutaraldehyde as crosslink agent. To trap the drug inside hydrogel
network, two methods has been used, those are post loading method and in situ loading method. Both methods has
been tested with drug entrapment efficiency test and drug dissolution method. The data obtained is used for analyzing
drug dissolution mechanism with mathematical model of drug release mechanism as a parameter and also use both
Scanning Electron Microscope (SEM) and optical microscope to identify interconnection pore inside and on the
surface of the hydrogel.

MATERIAL AND METHODS

The entire experiment of this study was conducted by in vitro study. The material used in this research among
others is amoxicillin trihydrate from PT. Kimia Farma, generic tablet and tablet patent (Amoxsan®) of amoxicillin
trihydrate, chitosan with DA 94.13% (medical grade) and 25.20 cps viscosity from PT. Biotech Surindo, methyl
cellulose from Sigma-Aldrich, Glutaraldehyde, glacial acetic acid pro analysis from Merck, and CaCO3 as pore
forming agent.

Preparation of the Hydrogel

Methyl cellulose (60:40 ratio w/w to the weight of chitosan) was added to 4 g of chitosan that has been dissolved
into 100 mL of 2% acetic acid in a beaker glass. Then the beaker glass was placed on a hot plate with magnetic stirrer.
The crosslink agent, glutaraldehyde was added (2% w/w against the total weight of monomer) after system become
homogenous and stirred for 3 hours. After glutaraldehyde blend in with the viscous solution of hydrogel, then add
CaCO3 as pore forming agent (15% w/w against the total weight of ingredients used) and stirred it until become a
homogenous solution. Finally, the solution was poured into a mold to form the hydrogel film and dried it in an oven
for 24 hours at a temperature of 60 ºC8.

Determination of Swelling Ratio

Swelling ratio was determined by immersing the hydrogel into water (aquabidest) for 30 minutes at room
temperature. Then the hydrogel surface was blotted using soft paper. The swelling ratio of hydrogel was determined
by the equation:
Wk−Wa
Swelling ratio (%) = 𝑥 100% (1)
Wa
Wk is the weight of hydrogel after swelling at a predetermined time (t), and Wa is the weight of hydrogel before
immersion4.

Determination of Floating Time and Floating Lag Time

Floating lag time is the time required for the hydrogel to be raised to the surface of the medium and be float while
floating time is how long hydrogel floats on the surface of the medium. Determination of floating time and floating
lag time were measured in a vial containing 10 mL of pH 1.2 solution at 37 ± 0.5 °C through visual observation4.

Encapsulation of Amoxicillin Trihydrate (In Situ Loading Method)

Encapsulation of amoxicillin trihydrate by in situ loading method was applied to optimum hydrogel (produce the
fastest floating lag time and floating time more than 3 hours). Preparation of optimum hydrogel which loaded by
amoxicillin trihydrate using the same procedures as in the preparation of hydrogel, but there was the addition of
amoxicillin trihydrate (500 mg) before the addition of 2% glutaraldehyde.
 Encapsulation of Amoxicillin Trihydrate (Post Loading Method)
The optimum hydrogel was immersed into 50 mg/mL amoxicillin trihydrate solution (the amount of solution used
accordance with the swelling ratio of optimum hydrogel) for 1 hour. Then, the hydrogels dried into the oven at a
temperature of 40 °C until a constant weight is obtained.

Determination of Encapsulation Efficiency of Amoxicillin Trihydrate

The hydrogel (mass already known) was immersed into 5 mL of pH 1.2 solution and sonicated for 1 hour. 1 mL
filtrate was taken and diluted to 10 mL; then the filtrate was analyzed using UV-Vis spectrophotometer at the
maximum wavelength of amoxicillin trihydrate9.

Dissolution Test of Amoxicillin Trihydrate

The hydrogel (mass already known) was immersed into 10 mL of pH 1.2 solution and shook using shaker (100
rpm at 37 ± 0.5 °C ). 1 mL filtrate was taken each interval (0, 5, 10, 20, 30, 60, 90, 120, 150, 180) minutes, and diluted
to 10 mL then analyzed the filtrate using UV-Vis spectrophotometer at the maximum wavelength of amoxicillin
trihydrate.

Characterization of Hydrogel by FTIR Instrument

The hydrogel characterized by FTIR and compared to the others. A thin sheet of sample hydrogel placed on a
holder which contain KBr powder. The transmittance was measured at wavenumber 400-4000cm-1.

Characterization of Hydrogel by Scanning Electron Microscope and Optical Microscope

The morphology of hydrogel was observed by both microscope. A thin sheet of sample hydrogel placed in a
preparation container, then manually set up the focus of the optic and the intensity of the light. For observing with
SEM, preparation have the same step with preparation using optical microscope, the difference take place before
sample placed inside the chamber, the sample is have to get to coated first using some element that can conduct
electron well, such as gold.

RESULT AND DISCUSSION

Hydrogel Characterization Using FTIR

The FTIR spectrum of hydrogel that has been mixed with pore forming agent shows the functional groups that it
has, is not much different with the functional groups contained on FTIR spectrum of hydrogel without pore forming
agent. The differences lies on some wave number, such as in wave number 3550 cm-1 at hydrogel with CaCO3 have
wide absorption came from O-H stretch vibration, and also shifting wave number is occurred to C=O stretch vibration
which originally take place in wave number 1700 cm-1, after pore forming agent was added the wave number shifted
to 1697 cm-1. New absorption band was detected in 877.65 cm-1 which is adsorption from carbonate group (Miller &
Wilkins, 2012), that means CaCO3 as pore forming agent has been successfully trapped inside chitosan-methyl
cellulose hydrogel network.
 Hydrogel+PFA
Hydrogel

Figure 1. Compared FTIR Spectrum between Chitosan-Methyl Cellulose Hydrogel with and without CaCO3
as Pore Forming Agent

Hydrogel Swelling Test

Hydrogel without pore forming agent has biggest swelling ratio because inside the polymer network there is no
other particles causing hydrogel can expand freely when absorb water. However, hydrogel with in situ loading
entrapment method have bigger swelling ratio compared to hydrogel with post loading entrapment method. This
difference caused by drug position when entrapped inside hydrogel, in in situ loading method the drug mixed
throughout polymerization reaction occurs, and it lead to drug distribution is between the polymer network causing
the network is more fragile and when hydrogel absorb water, it expand more than without drug or using post loading
method.

Swelling Ratio of Chitosan-Methyl

Cellulose Hydrogel
672%

459%

230%
172%

C-MS Hydrogel C-MS C-MS Hydrogel C-MS Hydrogel In

Hydrogel+PFA Post Loading Situ Loading

Figure 2. Compared Swelling Ratio of Chitosan-Methyl Cellulose Hydrogel

Chitosan-Methyl Cellulose Hydrogel Floating Lag time & Floating Time

Chitosan-Methyl Cellulose Hydrogel without drug inside polymer network causing it have fastest floating lag time,
and there are two big factors involved. First, the mass difference between hydrogel with drug trapped inside and
hydrogel without drug inside the network. Amoxicillin trihydrate has a molar mass of 419,449 gr/mol that make the
particle quite big and give an impact to the hydrogel mass and density, affecting it floating lag time properties. The
second factor is, the drug particle which is larger than pore forming agent particle can inhibit the interaction occurs
between acid solution and pore forming agent, causing pore forming agent harder to react and producing CO 2 that
make longer time to float.
Table 1. Compared Result of Chitosan-Methyl Cellulose Hydrogel Floating Lag time and Floating Time Test
Sample Floating Lag Time Floating Time
Hidrogel C-MS 11 minutes 24 seconds >180 minutes
In Situ Loading 13 minutes 50 seconds >180 minutes
Post Loading 17 minutes 4 seconds >180 minutes

Drug Entrapment Efficiency Test of Amoxicillin Trihydrate

Based on Figure 3. The result of Drug entrapment Efficiency Test shows that amoxicillin Trihydrate that entrapped
by in situ loading method has higher percentage than using post loading method. The different is significant, for in
situ loading method it has 100% while the post loading method has only reach 72%. The different percentage is caused
by when the drug get entrapped, for in situ loading method the drug get entrapped when polymerization reaction
occurs, means the drug is already inside the polymer network when molding process take place and it signify there
are no drug particles is being wasted. Still, for post loading method, the drug entrapment occurs when hydrogel has
been molded and shaped, causing the entrapment process is harder to be done.

Drug Entrapment Efficiency of Amoxicillin

Trihydrate in Chitosan-Methyl Cellulose
Hydrogel
Effciency Percentage (%)

100
90
80
70
60
50
post loading in situ loading

Figure 3. Percentage result from Drug Entrapment Efficiency of Amoxicillin Trihydrate using Post Loading
Method and In Situ Loading Method

Drug Dissolution Test of Amoxicillin Trihydrate Encapsulated by Hydrogel

As seen on figure 4. the result from drug dissolution test of amoxicillin trihydrate shows that with in situ loading
method, the drug dissolution belongs to controlled release category while post loading method indicate otherwise. In
situ loading method can create controlled release dissolution because of the drug position inside polymer network is
distribute equally and it’s the cause of the drug was added when hydrogel still in viscous phase. In the last time interval
(180 minutes) it can be seen that accumulative percentage of amoxicillin trihydrate dissolution for in situ loading
method reach value of 96%, indicates almost all of the drug contained inside the hydrogel is released. However with
post loading method, in the same time interval, its only reach value of 72% and it was the effect from interaction that
occurred between the drug and hydrogel network when the hydrogel is already in solid phase.
 Drug Dissolution Test of Amoxicillin
Trihydrate

Accumulative Percentage (%)

100
80
60
40 post loading
20 in situ loading
0
0 50 100 150 200
TIme (minutes)

Figure 4. Accumulative Percentage from Drug Dissolution Test of Amoxicillin Trihydrate Encapsulated by
Chitosan-Methyl Cellulose Hydrogel

Drug Release Mechanism of Amoxicillin Trihydrate Encapsulated by Chitosan-Methyl

Cellulose Hydrogel
By using four mathematical models of drug release as parameter, and analyzing the data obtained from drug
dissolution test it shows that drug dissolution mechanism of amoxicillin trhydrate from chitosan-methyl cellulose
hydrogel by both method post and in situ loading method have the same result. Both method indicates drug dissolution
mechanism closing to the value of Higuchi drug dissolution model, signify that using either post loading method or in
situ loading method the drug will come out from hydrogel network by diffusion. Based on Fickian’s law, dissolution
of amoxicillin trihydrate from hydrogel network is occurred when the dissolution media penetrate into hydrogel
network, dissolve and carry the dissolved drug out from the network via hydrogel’s pores 11. Since the drug release
mechanism have the same result on both method, the next step is to compare Higuchi constant (KH) between using the
Higuchi equation and the equation obtained from plotting between accumulative dissolution value and square root
value of time interval.
1
𝑄 = 𝐾𝐻 𝑡 ⁄2 (Higuchi Equation)
1
Where Q is accumulative value of drug dissolution in units of time, KH is Higuchi constant and 𝑡 ⁄2 is square root
of time. While the KH value from linear function of plotted graphic obtained from equation 𝑦 = 𝑎 + 𝑏𝑥 and KH=b.
The result from post loading method is KH from graphic have value of 4.48 while from Higuchi equation have value
0.34. The same result have obtained from in situ loading method, K H value from graphic is 5.94 and KH value from
Higuchi equation is only 0.65. the significant differences between K H value obtained from graphic and equation
indicates that the drug release mechanism only follows the Higuchi model in some point of time interval.

Tabel 1. r2 value from each Mathematical Drug Release Mechanism Model of Amoxicillin Trihydrate

nilai regresi (r2)

Metode
Orde Nol Orde Satu Higuchi Korsmeyer-Peppas
in situ loading 0.7498 0.8854 0.9089 0.8525
post loading 0.8976 0.9345 0.9367 0.7342

Even though the interaction occurred between chitosan and methyl cellulose as hydrogel network is strong enough,
but because the presence of pores inside and on the surface of hydrogel causing the drug go out from the hydrogel by
diffusion trough existing pores without having to wait the network to degraded by acid solution. The interconnection
in the polymer network is exist and amoxicillin trihydrate using diffusion as drug release mechanism is proved by the
result from SEM and optical microscope characterization in figure 5. and 6.
 Figure 5. Imaging of Chitosan-Methyl Cellulose Hydrogel Entrapping Amoxicillin Trihydrate using Optical
Microscope with Magnification of 40x
The morphology of hydrogel with post loading method is smoother and the pores existed is bigger compared to
hydrogel with in situ loading method. This different size of pore is the reason why the drug release category of post
loading method is included in quick release category while in situ loading is in controlled release category. Beside the
impact of the pore’s size, the degradation of hydrogel network allows pore located inside hydrogel network connected
to the other and creating interconnection. This phenomenon causes the drug release is harder to control and the drug
release process become quick release, yet the presence of interconnection pores causes the drug entrapped in the
middle of hydrogel network easier to reach by dissolution media and make the drug dissolution more optimal.
 Figure 6. Cross Section Imaging of Chitosan-Methyl Cellulose Hydrogel Entrapping Amoxicillin Trihydrate
using Scanning Electron Microscope (SEM) with different time interval (a) 0 minute (b) 60 minutes (c) 180
minutes (left) In Situ Loading (right) Post Loading
It can be seen at figure 6. using Scanning Electron Microscope (SEM) that the interconnection pore is
occurred inside hydrogel network before it submerged with acid solution indicating the interconnection is created
when undergo the molding process. The main factor why interconnection pore is occurred is when pore forming agent
which is CaCO3 added to the mixture of hydrogel, the particles are not dissolve well in the mixture cause there is some
particle that big enough to produce CO2 since the hydrogel mixture contain acid solution from acetic acid. Drug
dissolution Test result shows that in situ loading method is more effective than post loading method, because in the
last time interval only accumulative percentage of drug dissolution from in situ loading method that have value
approaching 100%. However, in situ loading method have a weakness that is the drug entrapment method affecting
physical properties of the hydrogel in surface morphology and the strength of the network.

CONCLUSION
Chitosan-methyl cellulose hydrogel as drug dosage of Floating Drug Delivery System in controlled release
category is more effective using in situ loading as drug entrapment method. Drug release mechanism from hydrogel
network with post loading and in situ loading method have the same result which is approaching to Higuchi drug
release model indicates drug release mechanism follows diffusion technique. Polymer network undergo degradation
process caused by interaction between hydrogel and acid solution, but doesn’t affect drug release mechanism.
Hydrogel network with in situ loading method having damaged more than post loading method. The size of
interconnected pore affected by drug entrapment method and the size particle of drug and pore forming agent itself.
Dissolution process also make pores in hydrogel network to create interconnection, and it proved by imaging results
from optical microscope and SEM. Drug entrapment method doesn’t have big impact to drug release mechanism,
since both method have the same result.

ACKNOWLEDGEMENT
The Authors would like to thank Directorate of Research and Community Engagement (DRPM) Universitas
Indonesia for supporting our research financially through Hibah Publikasi Internasional Terindeks untuk Tugas Akhir
(Hibah PITTA) 2017. We also would like to thank Department of Chemistry, Universitas Indonesia for supporting us
and providing the instrumental facilities for our research. Emil Budianto supervised this research and designed the
research pipeline, while Anastasya conducted the experimental details, analyzed the data, and wrote the final
manuscript. Herewith, we would declare that there is no conflict of interest regarding this manuscript.

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