1. INTRODUCTION:
Hydrogels are three dimentional-netwok of hydrophilic
polymer that can swell in aqueous solution without dissolving or losing their
structural integrity. The network is often formed by covalently cross-linked
polymers. On the other hands, water swellable materials can also results from
ionic bonds, physical entanglements, crystallites, hydrogen bonds and Van der
Waals force In the swollen state they are soft and rubbery, resembling to
living tissue, exhibiting excellent biocompatibility1. For these
biocompatibility properties hydrogel has a wide application in the medical, pharmaceutical and related field such as wound
dressing, contact lenses, artificial implants and drug delivery system2-7.
Due to lack of control drug release from conventional
drug formulations in response to physiological requirements have led to
development of controlled drug delivery system Recently hydrogel become
important as a matrix for controlled release. Many synthetic and naturally
derived materials have been reported to form well characterized hydrogels.
Among this materials gelatin has attractive feature as the staring material for
biodegradable, non carcinogenic and hydrophilic7 biopolymer
containing large number of functional groups.
Gelatin readily under goes chemical cross linking,
which is very important for its use as a biomaterial. For this advantages
gelatin containing hydrogel has large application in field ranges from tissue
engineering 8 to drug delivery, gene therapy 9-12 and
wound dressing 13. The other component chosen in this work for
hydrogel preparation are acrylamide (AM) and 2- acrylamido-2-methyl propane
sulfonic acid (AMPS). AM is most widely commercially application water soluble
product14. There uses ranges from paper manufacture, water
treatment, through oil recovery, to soil modification and medical applications.
AM and AMPS based polymer have environmentally sensitive properties 15.
AMPS is ionic monomer and the polymer prepared from it has kwon for
hydrophiliciy, thermal stability, stability over broad pH range, ionic
character. These hydrogels have attractive application as wound dressing
materials since it adheres to healthy skin but not to wound surface and is
easily replaceable without damage to the heal wound16. In present
study we had taken tetracycline (TC) as model drug because it is one of the most
well known antibiotics and is very effective against many anaerobic microbes
associate with various periodontal disease involving both adult and juvenile
periodentitis patients17.
In this study, the synthesis of AMPS-Na, gelatin, AM
containing hydrogel by free radical polymerization method, its water sorption
behavior at different pH, temperature, salt concentration, its water diffusion
process and its in-vitro release properties at different pH are reported.
2. MATERIALS AND METHODS:
2.1.
Materials:
2- acrylamido-2-methyl propane sulfonic acid sodium
salt (Na-AMPS) was obtained by neutralization of 2- acrylamido-2-methyl propane
sulfonic acid (Vinati organic Ltd, Maharastra, India) with NaOH solution.
Acrylamide (AM) (Merk, Mumbai,India) was purified by recrystallizing twice
with ethanol. Gelatin and potassium peroxodisulfate (KPS) were purchased from
Merk (Mumbai, India). N,N'-Methyline bisacrylamide (MBA) was purchased from
Central drug house (P) Ltd (Mumbai, India). Tetracycline was purchased from
Merk (Mumbai, India). All other chemicals used were of analytical grade and
double distilled water was throughout the experiment.
2.2
. Preparation of
hydrogels:
The hydrogels were prepared by free radical
polymerization method as described by other worker18. In present
study 0.25 g of gelatin was dissolved in 5 ml of double distilled water. To
this solution 14.06 mM AM, 4.68 mM Na-AMPS, 0.073 mM initiator (KPS) and 0.12
mM crosslinker (MBA) were mixed by stirring vigorously. The whole mixture was
transferred into PVC straw and polymerization was carried out at 60ºC. After
complete polymerization the hydrogel was dried at 50ºC for 8 hrs. The hydrogels
were cut into small pieces and allowed to equilibrate for 7 days by changing
the swelling medium every day, so that any unreacted monomer may leach out.
After 7 days the gel were taken out from the swelling medium and air dried. To
understand the effect of the monomer composition on the swelling a series of
different composite hydrogels were prepared. The various composition of monomer
in preparation of hydrogels are given in the table I.
2.3.
The
characterization of hydrogels:
To characterize structural feature of prepared hydrogel
and also to know stability of drug in hydrogel, IR spectra of AMPS-Na, AM,
gelatin, TC, gel and composite gel were recorded on FT IR spectrophotometer
(Shimadzu 8400S ). The morphological feature of the prepared hydrogel was
investigated by using SEM (Carllessleo-1430 VP). Thermal properties of the
prepared hydrogels were evaluated by recording thermogravimetric analysis (TGA)
thermograms between 0 to 700ºC under N2 atmosphere and at a heating
rate of 10ºC/ min on TGA ( METTLER TOLEDO, STARe SW 9.10).
2.4. Swelling studies:
The progress of the swelling process was monitored
gravimetrically as described by other workers 19. In brief 40 mg of
dry hydrogel was allowed to equilibrate at definite volume of bidistilled
water. In every 30 min interval of times the hydrogel was taken out soaked
between two filter papers and finally recorded the weight. The swelling ratio
is calculated by the following equation
Experiments were repeated in triplicate for each gel
specimen and mean value was obtained.
2.5. Loading of drug:
Drug loading was carried out by allowing a preweighted
dry piece of hydrogel (40 mg) to swell in TC (5 mg/ ml) solution. After
equilibrium swelling, the hydrogel was taken out, dried in air and reweighed.
The percent of loading was calculated by following equation
Where Wl and Wd are weights (in
mg) of TC loaded and dry hydrogel respectively.
2.6
Release studies:
For the release studies, the dry drug loaded hydrogel
was immersed in to 50 ml of phosphate buffer (pH 7.4) at 300 C. At
regular interval of times, 5 ml of solution was withdrawn and equal amount of
the same dissolution was added back to the medium to maintain a constant
volume. The amount of drug release was determined by UV-vissible
spectrophotometer (Shimadu-1700) at 420 nm wave length. The results shown in
graph are mean of two determinations.
3.
RESULTS AND
DISCUSSION:
3.1. IR spectra studies:
The IR spectra of pure AM, Na-AMPS, gelatin and
prepared hydrogel are depicted in fig.1 (a-d). The IR spectra of the gel in
Fig. 1(d) clearly shows combined spectral feature of various functional groups
of AM, Na-AMPS, and gelatin. The peak at 3652 cm-1 is due to N-H
stretching of AM, Na-AMPS, gelatin. The peaks from 2788 to 3271 cm-1
for C-H (symmetric and asymmetric) stretching confirms the present of AM,
Na-AMPS in the network of polymer. The N-H bending and N-C stretching is
observed at 1525 cm-1 and 1222 cm-1 respectively. The C=O
stretching for AM, Na-AMPS is observed at 1695 cm-1. The
characteristic peak of Na-AMPS units can be seen at 1041 cm-1 due to
SO group20. The evidence of cross linker in he hydrogel is confirmed
by the peak at 678 cm-1 (secondary amide) for MBA.
Fig.1 IR spectra of AM (a), Na-AMPS (b), gelatin (c)
and prepared hydrogel (d)
The IR spectra of pure TC, blank composite hydrogel and
drug loaded composite gel are shown in figure 2 (a-c). In IR spectra of drug
loaded hydrogel all the peaks of blank composite gel are present but the peaks
of TC are hard to detect. The peaks 3627, 2931, 1697, 1533 cm-1 of
the blank composite hydrogels are slightly shifted to lower wave number at
3625, 2929, 1693 and 1527 cm-1 respectively in dug loaded gel. This
is may be due to intermolecular interaction of TC with gel network. No new
peaks are observed in IR spectra of TC load composite gel, this indicates that
there is no chemical interaction between TC and gel. From this observation it
can be say that the activity of the TC is not lose after loading.
Fig.2 IR spectra of TC (a), blank composite gel (b),
drug composite gel (c)
3.2
SEM analysis:
The morphological feature of the prepared hydrogel has
been investigated by recording SEM micrographs as shown in fig3 (a-b) at
different magnification. From the micrograph it is clear that the surface of
the hydrogel is heterogeneous in nature having some small pores of size ranges
from 153 nm to 239 nm. The porous nature may be due to grafting of polymer
chain to each other. The scanning electron micrograph of drug loaded sample (c)
shows that small spherical like tetracycline are dispersed on the large surface
of hydrogel.
3.3 TGA analysis:
The thermo gravimetric analysis (TGA)
thermograms are depicted in fig 4. The thermo gram shows three step weight
lose. The first weight lose occurs between 20 to 130ºC, second at 200 to 340ºC
and third at 360 to 520ºC. The first weight lose is due to the evaporation of
water molecule and degradation of functional groups such as amide, sulfonic,
amine of the macromolecular chain by dehydration and generation more
stable group. It is observed that in second step 28.713% weight is lose where
temperature range is usually corresponded to de-polymerization process. The
third weight lose is due to final degradation of hydrogel.
(a)
(b)
(c)
Fig.3 SEM microgram of gel of gel (a), (b), drug
loaded gel (c).
3.4 Swelling studies:
Drug release rates are influence by the equilibrium
water uptake of the hydrogels21. It is well known that the swelling
capacity of the hydrogel is related to its chemical architecture. Table 1
indicates the equilibrium swelling ratio of different composite hydrogels at
same pH (phosphate buffer 7.4) media and at 32ºC. When the concentration of
Na-AMPS varies in the range 4.68 mM to 14.04 mM in the feed mixture, the SR
initially increases but beyond 7.03 mM it drops. The initial rise in swelling
ratio was due to the hydrophilic nature of AMPS-Na. Water of hydration
increased with higher hydrophilic content in the polymer chain. But these
occurs to a certain limits, further rise in the concentration, formation of
dense network structure take place, which prevents the water molecule
penetration inside the network. This leads to decrease in the swelling ratio.
For the same reason similar type of result were observed on varying gelatin
content from 0.25g to 1.0 g. The SR initially increased and falls beyond 0.5g.
The continues reduction of SR was due to the formation of a tight network
structure, which hinders the mobilities of the polymer chains and minimizes
water molecule penetration into polymer as the amount of AM has increased from
3.51 mM to 28.1 mM in the feed mixture. The influence of crosslinker
(MBA) on SR has been studied and results were summarized in the table 1. The
decrease of SR when the concentration of MBA rise from 0.06 mM to 0.25 mM in
the feed mixture is due to formation of more tight crosslink structure with
higher crosslinked content in the polymer network. This is results gradually drops in SR.
Table 1: Equilibrium swelling ratio (SR) and swelling
exponent n for different composite gels
|
Na-AMPS
mM
|
AM
mM
|
Gelatin
g
|
MBA
mM
|
SR
|
|
4.68
|
14.06
|
0.25
|
0.12
|
64.8
|
|
7.03
|
14.06
|
0.25
|
0.12
|
72.4
|
|
9.36
|
14.06
|
0.25
|
0.12
|
63.1
|
|
14.04
|
14.06
|
0.25
|
0.12
|
62.4
|
|
4.68
|
7.03
|
0.25
|
0.12
|
78.6
|
|
4.68
|
21.09
|
0.25
|
0.12
|
60
|
|
4.68
|
28.12
|
0.25
|
0.12
|
66.3
|
|
4.68
|
14.06
|
0.5
|
0.12
|
82.7
|
|
4.68
|
14.06
|
0.75
|
0.12
|
66.5
|
|
4.68
|
14.06
|
1.0
|
0.12
|
61
|
|
4.68
|
14.06
|
0.25
|
0.06
|
123.8
|
|
4.68
|
14.06
|
0.25
|
0.19
|
40.8
|
|
4.68
|
14.06
|
0.25
|
0.25
|
31.1
|
The pH of the swelling medium plays an important role
on SR of the hydrogel especially to the hydrogel which component are
polyelectrolyte in nature. The effect of pH on swelling is investigated at pH
range from 2 to 11.The results shown in fig. 5 indicated that the hydrogel
was pH sensitive and an optimum SR is observed at pH 7.4. At low pH the amino
groups of polymer chain gets protonated and forms hydrogen bond between
functional groups of polymer chains. This is leading to polymer–polymer
interaction predominating over polymer- water interaction, causes the gel to
shrink.. Again above pH 8, there was an electrostatic interaction between the
functional groups due to ionization of these groups. The amino groups were
completely deprotonated and contributed to the loss of solubility of the chain
segments and also formed new cross-links by hydrogen bond22. These
results a compact arrangement of the chain and low chain relaxation, so, low
swelling is observed. However, at pH 7 because of zero net charge on the
gelatin chain an optimum SR is observed. Similar type of result is observed by
other worker18.
Fig.5 Effect of pH on SR
Fig.6 Effect of temperature SR
When the temperature of the swelling medium increases
from 10 to 45o C the SR also increases as shown in fig. 6. The
observed result is due to increase of segmental mobility of the network chain.
A remarkable change in the swelling is observed at different salt concentration
shown in fig 7. The reduce in the swelling ratio at NaCl concentration from
0.001 M to 0.1 M was due to decrease in osmotic pressure. Since, there is a
balance between the osmotic pressure and the polymer elasticity, which sets the
physical dimensions of the hydrogels23. The osmotic pressure is
results from a net difference in the concentration in the mobile ions between
the interior of the gel and the solution. Addition of the ions to the outer
medium probably reduces the osmotic pressure and this brings decrease in the
swelling ratio at high salt concentration.
Fig.7 Effect of ionic concentration on SR
The water transport mechanism through the hydrogel can
be understand by fitting dynamic swelling data of all samples to an empirical
equation24,25 given below.
Where, k is the rate constant, n is the swelling
exponent, Wt and W∞ are the water intakes by the
swollen gel at time t and equilibrium time respectively. The values of n for
different compositional hydrogels are presented in table 1, which shows that
the water sorption mechanism follows non-Fickian diffusion processes for all
composite hydrogels. When the concentration of AMPS-Na increased from 4.68 mM
to 14.04 mM the value of n decreased from 0.70 to 0.54. This is due to the
formation of rigid tight crosslinked network with high content of AMPS-Na which
slowed down the chain relaxation rate lower than water diffusion rate. Again
with increasing gelatin and MBA concentration from 0.25 to 1.0 g and 0.06 to
0.259 mM respectively, there is slightly shifting of relaxation controlled to
diffusion controlled take place due to the formation of rigid tight crosslinked
network with high content of gelatin and MBA, which slowed down the chain
relaxation rate lower than water diffusion rate. Variation of AM and pH the
water sorption mechanism remains anomalous, suggesting that the relative rates
of diffusion of water molecules and chain relaxation remain almost identical in
the studied concentration range.
3.5 In vitro release studies:
To understand the release of tetracycline from the prepared
hydrogel, in vitro release experiments were carried out by varying compositions
of hydrogel and pH of the release medium. The influence of Na-AMPS on the % of
cumulative release is investigated at a range from 4.6 mM to 14.04mM in the
feed mixture. The results are presented in the fig.8. The gradual decrease of %
of cumulative release was due to the formation of compact network structure of
macromolecular chain at high content of Na-AMPS, which result decrease in % of
cumulative release.
Fig.8 Effect of Na-AMPS on % of release
Fig. 9 Effect of AM on % of release
The effect of AM on release profile is investigated and
shown in fig.9. The increase % of release with increasing concentration from
10.54 mM to 28.12 mM is due to the hydrophilic nature of AM which enhances
greater hydration of network and greater % of release. When the amount of
gelatin content rises from 0.25 to 1.0 g, the % of release initially increases
but beyond 0.5g of gelatin content it falls as shown in fig.10. The initial
rise of release rate is due to greater hydration of network and fall is due to
formation dens network structure. The reduction of release rate (fig11) while
the concentration of MBA varies form 0.06 mM to 0.25 mM is due to formation of
more tightly crosslinked rigid network structure with higher amount of
crosslinking agent.
Fig.10 Effect of gelatin on % of release
Fig.11 Effect of MBA on % of release
Fig12 Effect of pH on the % of release
Effect of pH on release profiles are studied at pH 2, 4
and 7.±0.1 and compared in fig.12. The % of cumulative release was reduced as
rising the pH. At lower pH the amide groups of TC were protonated which cause
repulsion among the entrapped drug molecules within the gel network. This
widens the mesh size of the gel and facilitates faster release.
4. CONCLUSION:
In this study, Na-AMPS based hydrogels were prepared by
free radical polymerization method. The swelling of the prepared hydrogel is
affected by composition of the polymer such as Na-AMPS, AM, gelatin and MBA in
the polymeric networks. Swelling experiment shows that the hydrogels were
sensitive to external environment such as pH, temperature and ionic strength.
The diffusion exponent calculated from empirical equation indicated that the
water transport mechanism for all hydrogels followed non-Fickian nature of
diffusion. The results of in-vitro drug release experiments showed that all the
hydrogel has sustained release properties and the release rate depends on the
swelling of the hydrogels and pH of the release medium. The release of TC
abruptly changes when the release medium changes from neutral to acidic medium.
It is believed that this hydrogel could be potentially used for localized drug
delivery system.
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