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INTERNATIONAL JOURNAL OF<br />
RESEARCH ARTICLE<br />
PHARMACEUTICAL INNOVATIONS ISSN 2249-1031<br />
<strong>Formulation</strong> <strong>and</strong> <strong>Evaluation</strong> <strong>of</strong> <strong>Bioadhesive</strong> <strong>Gel</strong> <strong>Incorporated</strong> Amoxicillin<br />
Trihydrate Loaded Microspheres for Periodontal Therapy<br />
Palak V Patel*, Dhiren J Daslaniya, Upendra L Patel <strong>and</strong> Ragin R.Shah<br />
Arihant School <strong>of</strong> pharmacy <strong>and</strong> bio- research institute, Adalaj, G<strong>and</strong>hinagar<br />
ABSTRACT<br />
To formulate <strong>and</strong> evaluate bioadhesive gel incorporated Amoxicillin trihydrate loaded<br />
microspheres in order to sustain, localize <strong>and</strong> target drug action in periodontal pockets.<br />
Amoxicillin trihydrate was loaded in gelatine microspheres using glutaraldehyde cross<br />
linking. The microspheres were evaluated for % yield, % drug entrapment, particle size, drug<br />
release as well as by scanning electron microscopy (SEM) <strong>and</strong> Fourier transform infrared<br />
spectroscopy (FTIR). Effect <strong>of</strong> drug: polymer ratio, stirring rate, glutaraldehyde amount,<br />
continuous phase amount on particle size <strong>and</strong> drug entrapment was also investigated. The<br />
microspheres were incorporated into gel <strong>and</strong> evaluated for pH, viscosity, bio adhesive<br />
strength <strong>and</strong> drug release. % yield, % drug entrapment <strong>and</strong> particle size <strong>of</strong> optimized batch<br />
(F8) microspheres were 94.2%, 94.3%, 80.1µm, respectively. SEM showed spherical<br />
geometry <strong>of</strong> microspheres. The pH, viscosity <strong>and</strong> bioadhesive strength <strong>of</strong> bioadhesive gel was<br />
6.9, 4000cps, 6.5gm, respectively. Sustain release <strong>of</strong> Amoxicillin trihydrate over a 6hr period<br />
from the microspheres <strong>and</strong> gel was achieved. <strong>Bioadhesive</strong> gel loaded with microspheres <strong>of</strong><br />
Amoxicillin trihydrate is a localized delivery system for the treatment <strong>of</strong> infection in<br />
periodontal pockets.<br />
Keywords: gelatine microsphere, bioadhesive dosage form, Amoxicillin trihydrate,<br />
periodontal pocket, glutaraldehyde cross linking<br />
INTRODUCTION<br />
Periodontitis is an inflammatory disease<br />
caused by chronic bacterial infection <strong>of</strong> the<br />
gum <strong>and</strong> bone supporting the teeth [1, 2] .<br />
Antimicrobial agents such as<br />
mitronidazole, tetracycline <strong>and</strong><br />
Amoxicillin trihydrate are used to treat<br />
bacterial infections in periodontal diseases<br />
[3] . Due to their short half life, these<br />
medications have to be taken frequently to<br />
maintain the desired therapeutic effect.<br />
Extensive efforts have recently been<br />
focused on targeting the drugs to a<br />
particular region <strong>of</strong> the body for extended<br />
period <strong>of</strong> time, thus maximizing drug<br />
Volume 3, Issue 3, May − June 2013<br />
availability <strong>and</strong> minimizing dose<br />
dependent side effects.<br />
<strong>Formulation</strong> <strong>and</strong> development <strong>of</strong> a gel<br />
based topical dosage form for<br />
antimicrobial drug will be proved to be<br />
worthwhile like ability to deliver drug<br />
more selectively to a specific site,<br />
avoidance <strong>of</strong> gastro-intestinal<br />
incompatibility, providing utilization <strong>of</strong><br />
drugs with short biological half-life,<br />
*Corresponding Author<br />
Palak V Patel<br />
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Improving physiological <strong>and</strong><br />
pharmacological response <strong>and</strong> provide<br />
suitability for self medication [4] .<br />
The sustain release dosage form<br />
maintaining relatively constant drug level<br />
in the plasma by releasing the drug at a<br />
predetermined rate for an extended period<br />
<strong>of</strong> time. One such in Microspheres as<br />
carriers <strong>of</strong> drug become an approach <strong>of</strong><br />
sustain release dosage form in novel drug<br />
delivery system.<br />
To obtain a sustained <strong>and</strong> targeted<br />
delivery, drugs can encapsulated in<br />
microspheres, which are then formulated<br />
as a gel . To overcome problems<br />
associated with the conventional system,<br />
we aimed to develop a bioadhesive gel<br />
formulation which can sustain <strong>and</strong> localize<br />
the drug in the periodontal pockets for an<br />
effective treatment [5] .<br />
Amoxicillin trihydrate is a moderatespectrum,<br />
bacteriolytic, β-lactam antibiotic<br />
used to treat bacterial infection caused by<br />
susceptible microorganism. Amoxicillin<br />
trihydrate has short half life 61.3 mins.<br />
Local delivery <strong>of</strong> Amoxicillin trihydrate<br />
have negligible impact on the micro flora<br />
residing in the other region <strong>of</strong> body [6] . Oral<br />
use <strong>of</strong> Amoxicillin trihydrate is associated<br />
with side effects like gastrointestinal<br />
disturbance, nausea, vomiting <strong>and</strong><br />
diarrhea. Topical application <strong>of</strong> the<br />
Amoxicillin trihydrate prevents these side<br />
effects <strong>and</strong> <strong>of</strong>fers potential advantage <strong>of</strong><br />
delivering the drug at the site <strong>of</strong> action.<br />
MATERIALS AND METHODS<br />
Materials<br />
Amoxicillin trihydrate obtained as a gift<br />
sample from Apex pharma, Hyderabad.<br />
<strong>Gel</strong>atin was obtained as a gift sample from<br />
S.D Fine chemicals, Mumbai. other<br />
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reagents <strong>and</strong> solvents used were <strong>of</strong><br />
pharmaceutical or analytical grade. The<br />
equipment used was sonicator, UV<br />
spectrophotometer, FTIR, stage<br />
micrometer microscope, scanning electron<br />
microscope, pH meter, brook field<br />
viscometer.<br />
Methods<br />
Preparation <strong>of</strong> microspheres by<br />
emulsion cross linking method<br />
<strong>Gel</strong>atine were accurately weighed <strong>and</strong><br />
mixed in 10 ml <strong>of</strong> distilled water,<br />
preheated to 60ºc, followed by the addition<br />
<strong>of</strong> Tween 80 (0.1%w/v). To this, 1 g <strong>of</strong><br />
amoxicillin trihydrate was added <strong>and</strong><br />
thoroughly mixed to obtain a<br />
homogeneous solution. The mixture was<br />
maintained at 50ºc,<strong>and</strong> than added drop<br />
wise into 100 ml <strong>of</strong> liquid paraffin<br />
containing Span 80 (0.1 %w/v) preheated<br />
to 60 ºc at constant stirring with 3- blade<br />
stirrer in order to form w/o emulsion.<br />
Glutaraldehyde was added drop wise to the<br />
emulsion <strong>and</strong> stirring <strong>and</strong> stirred for 1 hr at<br />
room temperature to stabilize the<br />
microspheres. The mixture was then left to<br />
cool at between 5-10 ºc f 2or 30 min to<br />
enhance settling <strong>of</strong> the microspheres.<br />
Microspheres were collected by filtration<br />
using Whatman filter paper <strong>and</strong> washed<br />
with 3×10 ml <strong>of</strong> chlor<strong>of</strong>orm followed by<br />
2×10 ml <strong>of</strong> 5 %w/v sodium<br />
metabisulphite, dried at room temperature<br />
<strong>and</strong> transferred to glass vials [7] .<br />
Effect <strong>of</strong> process variables on<br />
microsphere properties.<br />
<strong>Gel</strong>atine microspheres were prepared at<br />
different stirring rates, cross linking agent<br />
(gluteraldehyde) amount, continues phase<br />
amount <strong>and</strong> with various drugs: polymer<br />
ratios as stated in Table 1<br />
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Table 1: Process variables<br />
S. N. Process variables Values<br />
1 Stirring rate (rpm) 500, 1000, 1500<br />
2 Cross linking agent amount (ml) 0.5, 1.0, 1.5<br />
3 Drug : polymer ratios 1:1, 1:2, 1:3, 1:4<br />
4 Continuous phase amount (ml) 100, 150, 200<br />
Working formula<br />
Table 2: Batches for selection <strong>of</strong> drug: polymer ratio<br />
Batch<br />
Drug : polymer<br />
ratio<br />
Glutaraldehyde<br />
amount (ml)<br />
Stirring rate<br />
(rpm)<br />
Continuous phase<br />
amount (ml)<br />
F1 1:1 0.5 500 100<br />
F2 1:2 0.5 500 100<br />
F3 1:3 0.5 500 100<br />
F4 1:4 0.5 500 100<br />
Table 3 : Batches for selection <strong>of</strong> glutaraldehyde amount <strong>and</strong> stirring rate<br />
Batch Drug: Polymer<br />
ratio<br />
Glutaraldehyde<br />
amount (ml)<br />
Stirring<br />
Rate (rpm)<br />
Continuous phase<br />
amount(ml)<br />
F5 1:3 1.0 500 100<br />
F6 1:3 1.5 500 100<br />
F7 1:3 0.5 1000 100<br />
F8 1:3 1.0 1000 100<br />
F9 1:3 1.5 1000 100<br />
F10 1:3 0.5 1500 100<br />
F11 1:3 1.0 1500 100<br />
F12 1:3 1.5 1500 100<br />
Table 4: Batches to determine effect <strong>of</strong> continuous phase amount on microspheres<br />
Batch<br />
Drug: Polymer<br />
ratio<br />
Glutaraldehyde<br />
amount (ml)<br />
Stirring<br />
Rate (rpm)<br />
Continuous Phase<br />
amount (ml)<br />
F13 1:3 1.0 1000 150<br />
F14 1:3 1.0 1000 200<br />
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<strong>Evaluation</strong> <strong>of</strong> microspheres<br />
Determination <strong>of</strong> percentage yield<br />
The prepared microspheres were collected<br />
<strong>and</strong> weighed. The measured weight was<br />
divided by total amount <strong>of</strong> all non-volatile<br />
components, which were used for the<br />
preparation <strong>of</strong> microspheres. The % yield<br />
was calculated following formula<br />
W Rec<br />
% yield = × 100<br />
Weight (drug + polymer)<br />
Where<br />
W Rec = Weight <strong>of</strong> microspheres recovered<br />
Determination <strong>of</strong> percentage <strong>of</strong> drug<br />
loading<br />
The percentage <strong>of</strong> Amoxicillin trihydrate<br />
loading in microspheres can be estimated<br />
using Eq1.<br />
L =<br />
Q m<br />
W m<br />
x 100 …(1)<br />
where, L is the loading (%) <strong>of</strong><br />
microspheres, Qm is the quantity<br />
Amoxicillin trihydrate present in Wm <strong>of</strong><br />
microspheres <strong>and</strong> Wm is the weight <strong>of</strong> the<br />
microspheres in grams.<br />
Determination <strong>of</strong> encapsulation<br />
efficiency<br />
The amount <strong>of</strong> Amoxicillin trihydrate<br />
encapsulated in the microspheres was<br />
determined using Eq 2.<br />
E =<br />
Q p<br />
Q t<br />
x 100 ….(2)<br />
Where E is encapsulation efficiency (%),<br />
Qp is the quantity <strong>of</strong> drug encapsulated in<br />
the microspheres (g), Qt is the actual<br />
quantity <strong>of</strong> drug used for encapsulation (g),<br />
<strong>and</strong> Qp is the product <strong>of</strong> drug content per<br />
gm <strong>of</strong> microspheres <strong>and</strong> yield <strong>of</strong><br />
microspheres (g).<br />
Fourier Transform Infrared<br />
Spectroscopy<br />
Drug polymer interactions were studied by<br />
FTIR spectroscopy. The spectra were<br />
recorded for pure drug <strong>and</strong> drug loaded<br />
microspheres using FTIR. A pellet <strong>of</strong><br />
approximately 1 mm diameter <strong>of</strong> drug was<br />
prepared by compressing 3-5 mg <strong>of</strong> the<br />
drug with 100-150 mg <strong>of</strong> potassium<br />
bromide in KBr press (Model M-15,<br />
Techno Search Instruments). The pellet was<br />
mounted in IR compartment <strong>and</strong> scanned<br />
between wave number 4000 – 400 cm-1<br />
using a Shimadzu Model 8400 FTIR.<br />
Particle size analysis<br />
The particle size analysis was carried out<br />
by using optical microscopy. The<br />
microspheres were analyzed for size <strong>and</strong><br />
size distribution by first dispersing them in<br />
20 % v/v isopropyl alcohol to avoid<br />
swelling, vortexing for 3 min <strong>and</strong><br />
ultrasonicating for 30 s before sampling.<br />
The particle size analysis was carried out<br />
by using optical microscopy. About 100<br />
microspheres were selected r<strong>and</strong>omly <strong>and</strong><br />
their size was determined by using optical<br />
microscope fitted with st<strong>and</strong>ard micrometer<br />
scale.<br />
Scanning electron microscopy<br />
The sample the scanning electron<br />
microscopy (SEM) analysis was prepared<br />
by sprinkling the microspheres on one side<br />
<strong>of</strong> the double adhesive stub. The stub was<br />
then coated with gold using Jeol JFC 1100<br />
sputter coater. The SEM analysis <strong>of</strong> the<br />
microspheres was carried out using Jeol<br />
JSM 5300, Japan. The microspheres were<br />
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viewed at an accelerating voltage <strong>of</strong> 15-20<br />
kV.heres in grams.<br />
<strong>Evaluation</strong> <strong>of</strong> in vitro release by static<br />
method<br />
Microspheres, equivalent to 10 mg <strong>of</strong><br />
Amoxicillin trihydrate, were accurately<br />
weighed <strong>and</strong> transferred to 250 ml conical<br />
flask containing 100 ml phosphate buffer<br />
(pH 6.8). The flask was kept in an<br />
incubator at 37 °C, 1 ml samples withdrawn<br />
at regular intervals <strong>and</strong>, after suitable<br />
dilution, the amount <strong>of</strong> drug released was<br />
determined using a spectrophotometer at<br />
227.72 nm. Following each sample<br />
withdrawal, 1 ml <strong>of</strong> phosphate buffer was<br />
added to the release medium to replenish it.<br />
Five minutes before each sampling, the<br />
flasks were gently shaken by manually<br />
whirling it clockwise (15 revolutions) to<br />
minimize any concentration gradient within<br />
the release medium. The microspheres were<br />
allowed to settle down <strong>and</strong> clear<br />
supernatant medium withdrawn for drug<br />
analysis. The sample was filtered <strong>and</strong> the<br />
microspheres collected were transferred to<br />
the dissolution flask. Similarly, the release<br />
<strong>of</strong> Amoxicillin trihydrate from gelatin<br />
microspheres was determined<br />
spectrophotometrically at 227.72 nm<br />
(Shimadzu 1800) [5, 8] .<br />
Preparation <strong>of</strong> gel<br />
Preparation <strong>of</strong> the carbomer gel was based<br />
on a previously reported composition <strong>and</strong><br />
method. Accurately weighed Amoxicillin<br />
trihydrate (100 mg) was added to 15 ml <strong>of</strong><br />
water in a beaker <strong>and</strong> stirred well to<br />
dissolve the drug; 400 mg <strong>of</strong> carbopol was<br />
dissolved in this drug solution. <strong>Gel</strong>atin<br />
microspheres loaded with 100 mg <strong>of</strong><br />
Amoxicillin trihydrate was added to the<br />
drug carbopol solution <strong>and</strong> mixed well.<br />
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Methyl hydroxybenzoate (0.15 %w/w),<br />
propyl hydroxybenzoate (0.05 %w/w) <strong>and</strong><br />
sodium metabisulphate (0.1 %w/w)) were<br />
then added to microsphere/drug/carbopol<br />
mixture, while stirring at 250 rpm, to obtain<br />
a homogenous mixture. Stirring was<br />
continued until a lump-free suspension was<br />
obtained. Thereafter, 0.33 ml <strong>of</strong><br />
triethanolamine was added to produce a gel.<br />
This was followed by the addition <strong>of</strong> a<br />
sweetening agent (saccharin sodium, 0.1<br />
%w/w) <strong>and</strong> more water to make up to 20 g<br />
<strong>of</strong> gel [9] .<br />
<strong>Evaluation</strong> <strong>of</strong> bioadhesive gel<br />
incorporated microspheres<br />
Surface pH <strong>of</strong> the gel:<br />
An acidic or alkaline formulation is bound<br />
to cause irritation on mucosal membrane<br />
<strong>and</strong> hence this parameter assumes<br />
significance while developing a<br />
bioadhesive formulation. A digital glass<br />
electrode pH meter was used for this<br />
purpose. pH was noted by bringing the<br />
electrode near the surface <strong>of</strong> the<br />
formulations <strong>and</strong> allowing it to equilibrate<br />
for 1 min [10] .<br />
Viscosity Study:<br />
Viscosity <strong>of</strong> gels was studied on Brookfield<br />
viscometer by using spindle number 7 at 4<br />
revolutions per minute at constant<br />
temperature [11] .<br />
Estimation <strong>of</strong> drug content in formulated<br />
gels:<br />
<strong>Formulation</strong>s containing 1 mg <strong>of</strong> drug was<br />
taken in 10 ml volumetric flask, dissolved<br />
in pH 6.8 phosphate buffer made up the<br />
volume to 10 ml with pH 6.8 phosphate<br />
buffer <strong>and</strong> then filtered. Absorbance values<br />
were measured at respective λmax (227.72<br />
nm) for drug. Concentrations <strong>of</strong> drug were<br />
calculated from the st<strong>and</strong>ard calibration<br />
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curve prepared in pH 6.8 phosphate buffers<br />
[12] .<br />
Bioadhesion study:<br />
In the present study, bovine cheek pouch<br />
was used as a model mucosal surface for<br />
bioadhesion testing. The bovine cheek<br />
pouch was procured from slaughter house,<br />
then excised <strong>and</strong> trimmed evenly from the<br />
sides. It was then washed in phosphate<br />
buffer (pH 6.8) <strong>and</strong> was preserved in the<br />
same or used immediately. The two sides <strong>of</strong><br />
the balance were balanced with a 5 g<br />
weight on right h<strong>and</strong> side. The bovine<br />
cheek pouch excised <strong>and</strong> washed was tied<br />
tightly with the mucosal side upwards using<br />
a thread over the protrusion in the rubber<br />
block which is covered with inert<br />
aluminium surface. The block was then<br />
lowered into the glass container, which was<br />
then filled with isotonic phosphate buffer<br />
(pH 6.6) kept at 37°C±1°C, such that the<br />
buffer just reaches the surface <strong>of</strong> mucosal<br />
membrane <strong>and</strong> keeps it moist. This was<br />
then kept below the left h<strong>and</strong> set up <strong>of</strong> the<br />
balance. The film was then glued at the<br />
border adhered to a aluminium surface<br />
hanging on the left h<strong>and</strong> side <strong>and</strong> the beam<br />
raised, with the 5 g weight removed on the<br />
right pan side. This lowered the aluminium<br />
surface along with the film over the<br />
mucosa, with a weight <strong>of</strong> 5 g. The balance<br />
was kept in this position for 8 min <strong>and</strong> then<br />
slowly water was added to the plastic<br />
container in the right pan by pipette. The<br />
addition <strong>of</strong> water was stopped as soon as<br />
the detachment <strong>of</strong> two surfaces was<br />
obtained. Weight <strong>of</strong> water was measured.<br />
The excess weight in the pan i.e. total<br />
minus 5 mg is the force required to separate<br />
the film from the mucosa. This gave the<br />
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bioadhesive strength <strong>of</strong> the formulation in<br />
grams [13] .<br />
<strong>Evaluation</strong> <strong>of</strong> in vitro release by dynamic<br />
method<br />
<strong>Evaluation</strong> in vitro release studies <strong>of</strong><br />
Amoxicillin trihydrate from the carbomer<br />
gel was carried out at 37 °C using<br />
phosphate buffer (pH 6.8) as the release<br />
medium. A glass tube <strong>of</strong> 10 mm diameter<br />
<strong>and</strong> 100 mm height was taken. One end <strong>of</strong><br />
the tube was closed using an egg membrane<br />
with the help <strong>of</strong> adhesive tape while the<br />
other end was kept open <strong>and</strong> used as drug<br />
reservoir compartment. <strong>Gel</strong> (1 g)<br />
containing Amoxicillin trihydrate was<br />
accurately weighed <strong>and</strong> transferred to the<br />
glass tube in a vertical position through the<br />
open end. The gel was gently pushed down<br />
to the surface <strong>of</strong> the egg membrane with the<br />
help <strong>of</strong> a stainless steel spatula to ensure<br />
that all the gel was in contact with the<br />
membrane. Phosphate buffer (2 ml, pH 6.8)<br />
was added to the reservoir compartment to<br />
wet the gel. The glass tube was placed in a<br />
beaker containing 100 ml <strong>of</strong> phosphate<br />
buffer (pH 6.8) such that that the egg<br />
membrane is just immersed in the<br />
phosphate buffer which acted as the<br />
receiving compartment. The receiving<br />
compartment was magnetically stirred (100<br />
rpm, Remi, India) at 37 °C. Samples (1 ml)<br />
were withdrawn from the receiving<br />
compartment at regular intervals <strong>and</strong> the<br />
amount <strong>of</strong> dicl<strong>of</strong>enac sodium released from<br />
the gel was determined using a<br />
spectrophotometer at 227.72 nm<br />
(Shimadzu1800). After each withdrawal <strong>of</strong><br />
sample, equal quantity <strong>of</strong> phosphate buffer<br />
was added to the receiving compartment to<br />
replenish it [14] .<br />
Release kinetics<br />
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Data obtained from in vitro release studies<br />
were fitted to various kinetic equations [15]<br />
to determine the mechanism <strong>of</strong> drug release<br />
from the microspheres. The kinetic models<br />
used were zero order equation, first order<br />
equation <strong>and</strong> Higuchi release using the<br />
following plots: Q t vs t, log (Q 0 -Q t ) vs t <strong>and</strong><br />
Q t vs square root <strong>of</strong> t, respectively; where<br />
Q t is the amount <strong>of</strong> drug released at time t<br />
<strong>and</strong> Q 0 is the initial amount <strong>of</strong> drug present<br />
in the microspheres. T further, to ascertain<br />
the mechanism <strong>of</strong> drug release, the first 60<br />
% <strong>of</strong> drug release was fitted to Korsmeyer-<br />
Peppas<br />
model (Eq 3).<br />
Mt/Ma = kt n …. (3)<br />
Where Mt/Ma is the fraction <strong>of</strong> drug<br />
released at time, t, k is the rate constant <strong>and</strong><br />
n is the release exponent. The n value is<br />
used to characterize different release<br />
mechanisms.<br />
RESULTS AND DISCUSSION<br />
Drug- excipients compatibility study<br />
FTIR spectrum <strong>of</strong> Amoxicillin trihydrate<br />
<strong>and</strong> drug loaded microspheres are shown in<br />
Figure 1 <strong>and</strong> 2 respectively. From the FTIR<br />
spectra <strong>of</strong> Amoxicillin trihydrate <strong>and</strong> drug<br />
loaded microspheres, it was found that drug<br />
<strong>and</strong> excipients are compatible with each<br />
other.<br />
Figure 1: FTIR spectra <strong>of</strong> Amoxicillin trihydrate<br />
Figure 2: FTIR spectra <strong>of</strong> physical mixture <strong>of</strong> Drug <strong>and</strong> excipients<br />
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<strong>Evaluation</strong> <strong>of</strong> microspheres for selection <strong>of</strong> drug: polymer ratio<br />
Table 5: Selection <strong>of</strong> drug: polymer ratio<br />
Batch Drug: Polymer Ratio %Drug Entrapment Particle Size(µm) % Yield<br />
F1 1:1 20±2.6 200±4.5 30±3<br />
F2 1:2 68±3.7 235±3.7 52±1.6<br />
F3 1:3 90±1.9 330±6.7 87±2.2<br />
F4 1:4 Lump formation - -<br />
‣ Drug: polymer ratio: As increase the<br />
polymer concentration, increase in<br />
particle size due to increase viscosity<br />
<strong>of</strong> emulsion medium is increased. Due<br />
to increase in viscosity, larger<br />
emulsion droplets which are difficult to<br />
break. But increasing the concentration<br />
<strong>of</strong> polymer, increase entrapment<br />
efficacy. Further increasing<br />
concentration <strong>of</strong> polymer, lump<br />
formation due to aggregates <strong>of</strong><br />
microspheres.<br />
‣ In F3 batch highest drug entrapment<br />
obtained. So, Drug: Polymer ratio 1:<br />
3 was selected. For reducing particle<br />
size <strong>of</strong> microspheres stirring rate &<br />
glutaraldehyde<br />
amount optimized.<br />
Table 6: Selection <strong>of</strong> glutaraldehyde amount <strong>and</strong> stirring rate<br />
Batch<br />
Drug: Polymer<br />
ratio<br />
Glutaraldehyde<br />
amount (ml)<br />
Stirring<br />
Rate (rpm)<br />
%Drug<br />
Entrapment<br />
Particle<br />
Size(µm)<br />
% Yield<br />
F5 1:3 1.0 500 92±2.4 155±3 85±2.4<br />
F6 1:3 1.5 500 91±4.8 170±1.9 90±1.9<br />
F7 1:3 0.5 1000 83±2.1 121±3.0 87±1.8<br />
F8 1:3 1.0 1000 94±3 80±0.9 94±2.6<br />
F9 1:3 1.5 1000 93±1.2 159±2 92±0.7<br />
F10 1:3 0.5 1500<br />
F11 1:3 1.0 1500<br />
F12 1:3 1.5 1500<br />
Fiber<br />
obtained<br />
Fiber<br />
obtained<br />
Fiber<br />
obtained<br />
Fiber<br />
obtained<br />
Fiber<br />
obtained<br />
Fiber<br />
obtained<br />
Fiber<br />
obtained<br />
Fiber<br />
obtained<br />
Fiber<br />
obtained<br />
‣<br />
‣ Glutaraldehyde amount: increasing<br />
amount <strong>of</strong> cross linking agent retards the<br />
drug release. Crosslinking agent hardens<br />
the microspheres because <strong>of</strong> which drug<br />
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Cumulative % Drug Release<br />
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release is decrease. Further increasing<br />
concentration, larger particle size due to<br />
adherence <strong>of</strong> excess cross linking agents<br />
on the surface <strong>of</strong> the microsphere.<br />
‣ Stirring rate: stirring speed was low<br />
(500 rpm), larger particle size obtained.<br />
Due to inadequate stirring speed which<br />
was not able to break the emulsion<br />
droplets. At higher speed, fiber<br />
obtained 15 .<br />
‣ From these results, F8 batch in which<br />
stirring speed 1000 rpm <strong>and</strong><br />
glutaraldehyde amt is 1 ml.these<br />
microspheres had highest % drug<br />
entrapment <strong>and</strong> lowest avg particle size.<br />
So, F8 batch selected.<br />
Table 7: Effect <strong>of</strong> continuous phase amount on microspheres<br />
Batch<br />
Continuous phase<br />
amount (ml)<br />
Avg. particle size<br />
(µm)<br />
% Drug entrapment efficacy<br />
F13 150 160±3 62±3.6<br />
F14 200 155±1.9 57±2.7<br />
Values are means ± SD,( n=3).<br />
‣ Volume <strong>of</strong> continuous phase: Increase<br />
volume <strong>of</strong> continuous phase, emulsion<br />
droplets moved freely in medium, thus<br />
reducing collosion induced aggregates<br />
which could be the reason <strong>of</strong> high drug<br />
extraction in processing medium.<br />
Resulting in lower entrapment efficacy.<br />
Drug release study <strong>of</strong> microspheres (Batch F5 to F9)<br />
120<br />
100<br />
80<br />
60<br />
40<br />
20<br />
F5<br />
F6<br />
F7<br />
F8<br />
F9<br />
0<br />
0 100 200 300 400<br />
Fig 3: Cumulative Percentage drug released Vs Time<br />
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In F8 batch 96.0% cumulative drug release<br />
obtained within 6 hrs so F8 batch is<br />
selected for incorporation in to gel. F8<br />
batch microspheres had smallest particle<br />
size <strong>and</strong> highest % drug entrapment. So, F8<br />
batch is selected.<br />
Scanning electron microscope<br />
Scanning electron microscopy (Fig 4)<br />
confirmed that prepared Microspheres were<br />
spherical in shape. Also average particle<br />
size <strong>of</strong> the prepared micro particles was<br />
within the size range <strong>of</strong> 50 to 90 µm.<br />
Table 8: <strong>Evaluation</strong> <strong>of</strong> bioadhesive gel<br />
incorporated amoxicillin trihydrate microspheres<br />
<strong>Evaluation</strong><br />
Observation<br />
parameters<br />
Surface pH 6.9±0.1<br />
Viscosity<br />
4000±0.09<br />
(centipoises)<br />
<strong>Bioadhesive</strong> strength 6.50±0.100<br />
(g)<br />
Drug content (%) 99.25±0.03<br />
Fig 4: SEM photographs <strong>of</strong> gelatin<br />
microspheres loaded with Amoxicillin<br />
trihydrate<br />
IN-VITRO DIFFUSION STUDY<br />
‣ In-vitro diffusion study <strong>of</strong> bioadhesive gel incorporated Amoxicillin trihydrate loaded<br />
microspheres (Batch F8) <strong>and</strong> compared with microspheres (Batch F8).<br />
120<br />
100<br />
80<br />
60<br />
40<br />
20<br />
Amoxicillin trihydrate<br />
microspheres in gel<br />
Amoxicillin trihydrate<br />
microspheres<br />
0<br />
0 100 200 300 400<br />
Time (min)<br />
Fig 5: In-vitro release <strong>of</strong> Amoxicillin trihydrate from gelatine microspheres <strong>and</strong><br />
microspheres-loaded gel.<br />
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The cumulative release <strong>of</strong> Amoxicillin<br />
trihydrate from microspheres <strong>and</strong> gel are<br />
shown in Figure 5. Drug from the<br />
microspheres was released in a controlled<br />
manner for ≥ 6 h. The release pattern was<br />
biphasic with an initial ‘burst’ release <strong>of</strong> 25<br />
% <strong>of</strong> the loaded drug was in the first 10<br />
min. Thereafter, release was slow but<br />
steady, <strong>and</strong> by the end <strong>of</strong> the 6 th hr, 96.1 %<br />
<strong>of</strong> the loaded drug was released.<br />
Amoxicillin trihydrate from the carbomer<br />
gel formulation is also shown in Figure 5.<br />
The release pattern was biphasic with an<br />
initial ‘burst’ release that was enhanced by<br />
the loading dose <strong>of</strong> Amoxicillin trihydrate.<br />
By the end <strong>of</strong> the first 10 min, 34 to 36 %<br />
<strong>of</strong> the drug was released; subsequently,<br />
release was teady <strong>and</strong> by the end <strong>of</strong> the 6 th<br />
hr, 95 % <strong>of</strong> the drug was released.<br />
RELEASE KINETICS<br />
The results <strong>of</strong> curve fitting into the<br />
mathematical models are given in Table 9.<br />
The results indicate the drug release<br />
behaviour from the formulated bioadhesive<br />
gel <strong>of</strong> Amoxicillin trihydrate.<br />
When the release rate <strong>of</strong> Amoxicillin<br />
trihydrate from the microsphere gel <strong>and</strong><br />
their respective correlation coefficients<br />
were compared, it was found to follow<br />
Higuchi model kinetics(R 2 =0.938)<br />
indicating drug release was diffusion<br />
controlled.<br />
Table 9: Results <strong>of</strong> curve fitting <strong>of</strong> in vitro drug release from bioadhesive gel containing<br />
microspheres.<br />
Zero<br />
<strong>Formulation</strong><br />
order<br />
First order Higuchi Korsmeyer pepps<br />
R 2 R 2 R 2 N<br />
<strong>Bioadhesive</strong> gel incorporated<br />
Amoxicillin trihydrate<br />
microspheres<br />
0.782 0.871 0.938 0.625<br />
For the tested formulation, the values <strong>of</strong> n<br />
on fitting the simple power equation<br />
Mt/M∞ = Kt n were found 0.625 for the<br />
release <strong>of</strong> Amoxicillin trihydrate from the<br />
bioadhesive gel, indicating anomalous (non<br />
fickian) diffusion, where drug release is<br />
controlled by combination <strong>of</strong> diffusion <strong>and</strong><br />
polymer chain relaxation mechanisms.<br />
CONCLUSION<br />
The formulated Amoxicillin trihydrate<br />
loaded gelatine microspheres for sustain<br />
release was found to be potential <strong>and</strong><br />
effective in terms yield, encapsulation<br />
efficacy, particle size <strong>and</strong> in-vitro release<br />
characteristics. The gel formulation , which<br />
consisted <strong>of</strong> drug loaded gelatine<br />
microspheres, showed sustain release <strong>of</strong><br />
Amoxicillin trihydrate ≥ 6 hr, thus<br />
indicating their suitability for the sustained<br />
delivery <strong>of</strong> the drugs for the treatment <strong>of</strong><br />
infections in periodontal pockets. However,<br />
further studies, including clinical tests are<br />
required to confirm the gel’s therapeutic<br />
efficacy.<br />
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