Research Article

1. Microphase separation and Gelation of Methylcellulose in
the presence of Gallic acid and NaCl as an In situ gel-
forming drug delivery system
(Journal Club- I Presentation)
PRESENTED BY:-
Divya D Shukla
19MPHTCH009
M.pharm ( Batch 2019-21)
Department of Pharmaceutical Technology
GUIDED BY:-
Dr. Gayatri C Patel
Associate professor
Department of Pharmaceutical Technology
RPCP-Changa
CHAROTAR UNIVERSITY OF SCIENCE AND TECHNOLOGY
CHANGA 1

2. Article Information
Article Type Research Article
Journal Name AAPS PharmSciTech
Impact Factor 2.401
Authors Tanatchaporn Sangfai, Vimon Tantishaiyakul, Namon Hirun, and Lin
Li
Publisher Springer
Year of Publication 2016
Volume Volume 21
DOI 10.1208/s12249-016-0546-7
2

3. CONTENT :-
3
Introduction
Objective
Materials and Methods
Results and discussions
Author’s conclusion
My learnings

4. 4
• Methyl cellulose is
amphiphilic
polysaccharide,
contains hydrophilic
hydroxy group and
hydrophobic methoxy
group.
• It can undergo a
thermo-reversible
phase transition from
sol to gel in an aqueous
solution upon heating
above 60°C.
• Different molecules or
additives lower or raise the
gelation temperature of MC.
• Including inorganic salts that
cause different effects on the
gelation of MC depending on
the salt type and concentration
INTRODUCTION
• A salting-out effect is to
depress the gelation
temperature of MC, while
a salting-in effect is to
increase its gelation
temperature.
• These salting-in and salting-
out effects result in the
hydration and dehydration
of the MC molecules.
• These phenomena may
hinder or facilitate the
association of the
hydrophobic units of MC.

5. OBJECTIVE
5
The objective was to develop
an in situ gel forming
hydrogel of MC by adding
GA and/or GA/NaCl.
• The system may be
administered in its sol state
and used to fill a wound
site (a deep wound or an
irregular space) and then be
converted into a gel so it
will adhere to the wound
site.

6. MATERIALS
AND
METHODS
Materials Used
6
Sr num Chemicals Purchased from
1 MC
(MC was dried in a vacuum oven at 60°C
overnight to remove residual water and then
kept in a desiccator and used without further
purification.)
Shin-Etsu Chemical Co., Ltd.
(Japan).
2 Doxycycline (DX)
Sigma-Aldrich (St. Louis,
MO, USA).
3 Gallic acid
4 NaCl Lab-Scan, Bangkok, Thailand

7. Methods
7
Sample Preparation Evaluation of prepared sample In Vitro Release Study
Antimicrobial Assay
Dissociation or
degelation of MC
Thermal Study DSC Calorimetry

8. For the preparation of MC stock Solution
• Required weight of MC was dispered in
hot water with vigorous stirring.
• Remaining water was added and mixture
was put in ice until solution becomes
transparent,
• Solution was kept in a refrigerator at 5°C
overnight to completely dissolve .
For the preparation of NaCl/MC samples:-
• Appropriate amounts of NaCl were dissolved in
water.
• To each NaCl solution, an equal volume of the
cooling stock solution of MC (3%) was added
and the solution was thoroughly mixed to obtain
1, 2, 3, and 4% w/v of NaCl in the MC solution.
For the preparation of Gallic acid stock
solution:-
• Required amount of GA was dissolved in
deionized water.
To prepare various concentrations of GA in NaCl
and MC:-
• The required amount of NaCl was added into the
appropriate GA solutions.
• To each mixture, an equal volume of the cooling
MC stock solution was added to obtain 0.1%
GA/NaCl/MC, 0.2%GA/NaCl/MC,
0.3%GA/NaCl/ MC, and 0.4%GA/NaCl/MC,
respectively.
For loading of the drug DX,:-
• Appropriate amounts of DX and NaCl were dissolved in the GA solution.
• An equal volume of the cooled MC stock solution was added to this mixture
and was thoroughly mixed to obtain a 0.25% w/v DX in 0.3GA/4NaCl/MC
(0.25DX/0.3GA/4NaCl/MC).
• All prepared solutions were kept in the refrigerator before further studies
Sample preparation
8
For the preparation of GA/MC samples:-
• Appropriate volumes of the GA stock solution were diluted with appropriate
volumes of water.
• To each GA solution, an equal volume of cooled 3% MC stock solution was added.
• Each mixture was thoroughly stirred to obtain 0.1, 0.2, 0.3, and 0.4% w/v of GA in
MC solution.

9. • Test Tube Tilting Method Analysis
9
Evaluation of prepared sample
•The samples were heated from 24 to 68°C
in a temperature-controlled water bath at
increments of 2°C every 5 min.
Samples were allowed to
equilibrate for 5 min after
each increment, and phase
transition was assessed by
inverting the tube after the
incubation period.
•The temperature at which
the samples did not flow
was recorded as the sol-to-
gel transition temperature.

10. Rheological Measurements
• The rheometer with a geometry of parallel plates of 55 mm in
diameter and a gap between the plates of 0.5 mm was used.
• The cold sample solution was placed on the bottom plate at 10°C.
• A thin layer of low-viscosity silicone oil was placed on the peripheral
surface of the solution to avoid dehydration during the measurements.
• The temperature dependence of storage modulus and loss modulus
was investigated .
• The thermal cycle measurement was started with heating from 10 to
80°C and subsequently cooled from 80 to 10°C at the heating and
cooling rate of 1°C/min.
10

11. Micro-DSC Measurements:-
• Calorimetric experiments were performed with
a micro- DSC through a thermal cycle in which
the sample was first heated from 10 to 80°C and
subsequently cooled to 10°C.
• The sample was injected into a sample cell with
a cell volume of 0.5 mL, and the reference cell
was filled with deionized water. After the end of
each cycle, the sample cell was washed by
distilled deionized water.
• A water-water DSC test was run to confirm the
cleanliness of the sample cell.
Turbidity Study:-
• The turbidity measurement was carried on using a UV-
vis spectrometer with a temperature controller.
• A cold solution of the sample was placed in the cuvette,
and deionized water was used as the reference.
• The cuvette was covered with a plastic cap to avoid
evaporation.
• The transmittance was measured at 500 nm during a
thermal cycle from 10 to 80°C and subsequently cooled
to 1°C at the same scanning rate as for the micro-DSC
measurement of 1°C/min.
11

12. Gravitational Flow Simulation:-
• A glass slide was incubated at the required
temperatures of either 25, 32, or 37°C.
• A 1mL sample of the mixtures
(0.3GA/NaCl/)& (0.25DX0/0.3GA/4NaCl/MC)
from the refrigerator in a liquid form was
loaded onto the slide,
• The gravitational flow simulation was observed
at 25, 32, and 37°C by lifting the glass slide
after equilibration for 2 or 10 min.
• The time for the mixtures to flow to the
bottom was recorded.
Antimicrobial Assay
• The samples were determined for their antibacterial activity in
Muller-Hinton agar (MHA) plates.
• To determine any possible synergistic effect of
0.25DX/0.3GA/NaCl/MC, its antibacterial activity was
evaluated in comparison with 0.25DX/4NaCl/MC .In addition,
the antimicrobial assay of NaCl/MC, 0.25DX, and 0.3GA was
also determined to detect the effect of each component in the
mixture.
• Each standard paper disk (6 mm) was impregnated with 50 μL
of the samples in the sol state and then plated on an individual
MHA.
• After incubation at 35°C for 24 h, the zones of inhibition were
measured. The assays were performed in triplicate.
SEM Measurements:-
• The prepared samples were lyophilized, and the dried samples were coated with gold to increase conductivity before
scanning.
• The surface morphology was subsequently investigated using a Scanning Electron Microscope operating at 20.0 kV.
12

13. 13
In Vitro Release Study
A modified dialysis method was used to investigate
the in vitro release behavior of the DX and GA from
the hydrogel.
2 mL of
0.25DX/0.3GA/4NaCl/MC in the liquid state from the
refrigerator as well as the solutions of DX and
GA was placed in a dialysis bag.
The dialysis bag was incubated
in 100 mL of phosphate-buffered saline (PBS) of pH 7.4
which had been prewarmed to 37°C with gentle shaking at
75 rpm.

14. The concentration of GA and DX in the release
medium was determined using an HPLC with
UV detection at 267 and 347 nm, respectively.
The retention times of 2.7 and 8.7 min were detected for
GA and DX, respectively
14
At certain time points 2 ml of the
medium was removed and replaced by
2 ml of the prewashed fresh medium.

15. RESULTS AND DISCUSSION
15
• TEST TUBE TILTING METHOD:_
• TTM was initially performed to determine the approximate gelation temperature
• Based on the test tube tilting method, pure MC (0.5–2.0%) formed a gel at between 56 and 62°C.
• The mixture of MC and GA had a lower gelation temperature compared to the MC alone.
• 0.4% GA did not lower the gelation temperature to 37°C while increasing the GA concentration to 0.6% caused
precipitation.
• In the absence of GA, the mixtures of various concentrations of NaCl (1.0–4.0% w/v) and 1.5% w/v MC
produced a gel at 42°C. However, 0.2GA/ NaCl/MC, 0.3GA/3NaCl/MC, and 0.3GA/4NaCl/MC formed gels at
34, 36, and 34°C, respectively.
• Therefore, the ternary mixtures of GA/NaCl/MC at certain concentrations could be used to produce
thermoresponsive gels at body temperature.

16. • With regard to the thermogelation of MC, the endothermic peak was
described as a sol-to-gel transition temperature.
• At a low temperature, MC was hydrated and existed in a sol state. As
the temperature was increased, MC lost its water of hydration and the
formation of hydrophobic aggregations of MC occurred.
• This caused cloudiness in the phase transition and subsequently
resulted in a gel network structure. The destruction of the hydrogen
bonds between the MC and water molecules or the cage structures of
water was mainly attributed to the endothermic peak observed in the
heating process.
• Figure shows the DSC heating curves for MC, and the mixtures of 0.1–
0.3GA/MC, 1–4NaCl/MC, and 0.1–0.3GA/NaCl/MC.
• The endothermic peak shifted to the left or to a lower temperature with
increasing GA concentration.
• The sol-to-gel temperatures of MC, 0.1GA/MC, 0.2GA/MC, and
0.3GA/MC were 60.9, 58.4, 56.4, and 54.6°C, respectively.All these
temperatures were higher than the physiological temperature of 37°C.
• It was of observed that the heating peak area significantly decreased
with increasing GA.
DSC Calorimetry
16

17. • The endothermic peak also shifted to the lower temperature with increasing NaCl concentration. The sol-to-
gel temperatures of 1NaCl/MC, 2NaCl/MC, 3NaCl/MC, and 4NaCl/MC were 55.7, 51.9, 48.4, and 45.5°C,
respectively.
• With the addition of NaCl, the salt ions attracted water molecules to surround the salt molecules and caused a
salt-out effect that depressed the gelation temperature.
• The salt decreased the solubility of MC or the hydrogen bonds between MC and water were broken, and this
enhanced the hydrophobic aggregations of the MC resulting in a lower gelation temperature.
17

18. • In the presence of NaCl, more energy was required to break the
hydrogen bonds of the cage structures and those between the MC chains
and water molecules.
• More energy was consumed for the interactions of ions and water
molecules. Consequently, ΔH was positive and increased with increasing
amounts of added NaCl.
• In order to further lower the gelation temperature to the physiological
temperature, GA was added into NaCl/MC.
• The endothermic peaks of the mixtures of
0.1GA/NaCl/MC,0.2GA/4NaCl/MC, and 0.3GA/NaCl/MC were 42.0,
38.0,and 36.7°C, respectively.
• The mixture of 0.3GA/NaCl/MC was suitable for use as a drug delivery
system that needed to be a gel at the physiological temperature.
Thermal Study:-
18

19. • MC underwent the gel-to-sol transition upon cooling. In
cooling process, the dissociation or degelation of MC was
associated with the hydration of the hydrophobic aggregates
which became weakened.
• Water molecules could approach to the hydrophobic
portions of MC, and the cage structures were formed
between the water molecules and methyl groups of MC.
• The cooling process was exothermic due to the formation of
the cage structures. In the presence of GA or NaCl, the
formation of cage structures was more difficult than for the
pure MC system.
• This was due to the strong attractive forces between the
water molecules and salt ions or the hydrophilic molecules
of GA.
• Hence, the exothermic peaks of GA/MC, NaCl/MC, and
GA/NaCl/MC systems shifted to the lower temperature in
the cooling process. Consequently, the increase of GA or
NaCl concentration caused a decrease of the degelation
temperatures of the mixtures.
Dissociation or degelation of MC
19

20. Effect of NaCl/GA on Rheological Properties and
Turbidity
20
• Upon heating, the transparent samples became increasingly
turbid due to the hydrophobic aggregates of MC. This caused
a microphase separation of polymer-rich and polymer-poor
regions.
• In the subsequent cooling process, the turbidity
turned progressively clearer and became homogenous or
transparent again.
• In this study, 50% transmittance was
defined as the clouding point or gelation temperature during
the heating and degelation temperature during cooling.
• With the increase of NaCl, GA, or GA/4NaCl contents in
the MC, all methods showed a decrease of the sol-to-gel
temperature upon heating and a decrease of the gel-to-sol
temperature upon cooling.

21. Morphological Properties
• The morphology of the freeze-dried MC and the mixtures was
investigated using SEM.
• In a previous study, there was evidence that an increase in
crosslinking density caused a decrease in the pore size.
• Therefore, the mixture of MC with NaCl may increase the cross-
linked density and consequently reduce its pore size. GA may also
increase the cross-linked density of MC but by a lower amount than
NaCl.
• GA itself may act as a cross-linking agent of MC, but this probably
produced a lower density than that produced by the hydrophobic
association of MC alone
• The pore size of the individual mixture was different with the order
of 4NaCl/MC << 0.3GA/MC ≅ 0.3GA/4NaCl/MC << plain MC.
This may indicate the distinct effect of 0.3GA and 4NaCl on the
formation of the gel network structures of the 4NaCl/MC and
0.3GA/MC.
• The pore size of 0.25DX/0.3GA/4NaCl/MC was much smaller than
that of 0.3GA/4NaCl/MC. This demonstrated that DX may be able
to increase the cross-linked density of the mixture. 21

22. Antimicrobial Assay
• No antimicrobial activity of 4NaCl/MC was detected.
• The antimicrobial activity of 0.25DX and 0.25DX/4NaCl/MC was comparable.
• In addition, 0.3GA showed only slightly antimicrobial activity. GA caused a synergistic inhibitory effect to some
microorganisms when it was mixed with DX.
• In this study, the percentage increase of the inhibition zones was calculated as:-
%increase inhibition= b−a * 100
a
• where a and b were the inhibition zones in the absence and presence of GA, respectively.
22

23. Gravitational Flow Simulation
• The thermosensitive behavior of the mixtures of 0.3GA/4NaCl/MC and 0.25DX/0.3GA/4NaCl/MC when applied to the
wound skin was investigated using gravitational flow simulation at 25, 32, and 37°C. A glass slide was used in this study
due to its hydrophilic wettability that imitated a wounded skin.
• After equilibrating the mixtures at 25°C on the slide for either 2 or 10 min, the mixtures were flowed to the bottom within
3s.
• After equilibrating the mixtures on the slide at 32°C for 2 min, a weak gel was observed and both mixtures flowed to the
bottom in 1 min due to gravity. After equilibrating for 10 min, a stronger gel was observed and both the 0.3GA/4NaCl/MC
and 0.25DX/0.3GA/4NaCl/MC remained on the slides but had moved a little in 20 min after lifting the slide.
• The DX may slightly decrease the viscosity of the mixtures at 32°C, but both were able to be deposited on the normal skin
surface. After equilibrating the samples at 37°C for a short period of 2 min and the slide was then lifted to a vertical
position for 30 min, both 0.3GA/4NaCl/MC and 0.25DX/0.3GA/4NaCl/MC still remained at the same position.
• Therefore, the mixtures of loaded and unloaded DX were able to remain on the surface of the wounded skin.
23

24. In Vitro DX and GA Release
• As shown in Fig, the thermal and rheological properties of
0.3GA/4NaCl/MC and 0.25DX/0.3GA/4NaCl/MC were
comparable and this indicated that 0.25DX did not alter the
thermoresponsive properties of the 0.3GA/4NaCl/MC in the
temperature sweep experiment.
• Nevertheless, these two mixtures showed slight differences in
gel strengths at 32°C.
• The release of free DX and GA from the solution (85% in 3 h)
was faster than from the mixture (45 and 52% in 3 h,
respectively, and about 100% in 18 h). This indicated that the
dialysis bag had some retaining effect on the release of the
drugs.
24

25. Author’s conclusion
• NaCl lowered the gelation temperature of MC GA could also
lower the gelation/degelation temperature during heating/ cooling,
respectively.
• GA could interact with MC via hydrophobic interactions but not
NaCl. Both GA/MC and NaCl/MC mixtures could dissociate
water from their MC and induce a hydrophobic association with
MC.
• Due to the ability to interact with MC, the mixture with GA
showed a larger microporous morphology of its xerogel compared
to that of the NaCl/MC xerogel as observed by SEM.
• This indicated that the GA/MC mixture produced a lower density
of cross-linking than the NaCl/MC mixture, probably due to the
interaction between the GA and MC.
• MC in the presence of NaCl or GA reduced the pore size much
more than the plain MC. Therefore, both NaCl and GA could
promote the hydrophobic aggregation and increase the cross-
linking density of MC.
• GA could provide a synergistic inhibitory effect on various
bacteria when mixed with DX.
25

26. My learnings
26
• I learnt about the technique to reduce temperature of MC
using GA and NaCl and learnt about the incorporation of
antimicrobial agent Doxycycline in hydrogel.
• I learnt GA is able to facilitate the gelation of MC by
interacting with the water molecules, and decreases the
hydration of MC and decrease the gelation temperature
• I learnt about the effect of GA/NaCl on thermal behavior.
• I understood the methods of characterization of hydrogel.

27. 27
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