Formulation and Evaluation of Bioadhesive Gel Incorporated ... - IJPI

INTERNATIONAL JOURNAL OF 

RESEARCH ARTICLE 

PHARMACEUTICAL INNOVATIONS ISSN 2249-1031 

Formulation and Evaluation of Bioadhesive Gel Incorporated Amoxicillin 

Trihydrate Loaded Microspheres for Periodontal Therapy 

Palak V Patel*, Dhiren J Daslaniya, Upendra L Patel and Ragin R.Shah 

Arihant School of pharmacy and bio- research institute, Adalaj, Gandhinagar 

ABSTRACT 

To formulate and evaluate bioadhesive gel incorporated Amoxicillin trihydrate loaded 

microspheres in order to sustain, localize and target drug action in periodontal pockets. 

Amoxicillin trihydrate was loaded in gelatine microspheres using glutaraldehyde cross 

linking. The microspheres were evaluated for % yield, % drug entrapment, particle size, drug 

release as well as by scanning electron microscopy (SEM) and Fourier transform infrared 

spectroscopy (FTIR). Effect of drug: polymer ratio, stirring rate, glutaraldehyde amount, 

continuous phase amount on particle size and drug entrapment was also investigated. The 

microspheres were incorporated into gel and evaluated for pH, viscosity, bio adhesive 

strength and drug release. % yield, % drug entrapment and particle size of optimized batch 

(F8) microspheres were 94.2%, 94.3%, 80.1µm, respectively. SEM showed spherical 

geometry of microspheres. The pH, viscosity and bioadhesive strength of bioadhesive gel was 

6.9, 4000cps, 6.5gm, respectively. Sustain release of Amoxicillin trihydrate over a 6hr period 

from the microspheres and gel was achieved. Bioadhesive gel loaded with microspheres of 

Amoxicillin trihydrate is a localized delivery system for the treatment of infection in 

periodontal pockets. 

Keywords: gelatine microsphere, bioadhesive dosage form, Amoxicillin trihydrate, 

periodontal pocket, glutaraldehyde cross linking 

INTRODUCTION 

Periodontitis is an inflammatory disease 

caused by chronic bacterial infection of the 

gum and bone supporting the teeth [1, 2] . 

Antimicrobial agents such as 

mitronidazole, tetracycline and 

Amoxicillin trihydrate are used to treat 

bacterial infections in periodontal diseases 

[3] . Due to their short half life, these 

medications have to be taken frequently to 

maintain the desired therapeutic effect. 

Extensive efforts have recently been 

focused on targeting the drugs to a 

particular region of the body for extended 

period of time, thus maximizing drug 

Volume 3, Issue 3, May − June 2013 

availability and minimizing dose 

dependent side effects. 

Formulation and development of a gel 

based topical dosage form for 

antimicrobial drug will be proved to be 

worthwhile like ability to deliver drug 

more selectively to a specific site, 

avoidance of gastro-intestinal 

incompatibility, providing utilization of 

drugs with short biological half-life, 

*Corresponding Author 

Palak V Patel 

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Improving physiological and 

pharmacological response and provide 

suitability for self medication [4] . 

The sustain release dosage form 

maintaining relatively constant drug level 

in the plasma by releasing the drug at a 

predetermined rate for an extended period 

of time. One such in Microspheres as 

carriers of drug become an approach of 

sustain release dosage form in novel drug 

delivery system. 

To obtain a sustained and targeted 

delivery, drugs can encapsulated in 

microspheres, which are then formulated 

as a gel . To overcome problems 

associated with the conventional system, 

we aimed to develop a bioadhesive gel 

formulation which can sustain and localize 

the drug in the periodontal pockets for an 

effective treatment [5] . 

Amoxicillin trihydrate is a moderatespectrum, 

bacteriolytic, β-lactam antibiotic 

used to treat bacterial infection caused by 

susceptible microorganism. Amoxicillin 

trihydrate has short half life 61.3 mins. 

Local delivery of Amoxicillin trihydrate 

have negligible impact on the micro flora 

residing in the other region of body [6] . Oral 

use of Amoxicillin trihydrate is associated 

with side effects like gastrointestinal 

disturbance, nausea, vomiting and 

diarrhea. Topical application of the 

Amoxicillin trihydrate prevents these side 

effects and offers potential advantage of 

delivering the drug at the site of action. 

MATERIALS AND METHODS 

Materials 

Amoxicillin trihydrate obtained as a gift 

sample from Apex pharma, Hyderabad. 

Gelatin was obtained as a gift sample from 

S.D Fine chemicals, Mumbai. other 


reagents and solvents used were of 

pharmaceutical or analytical grade. The 

equipment used was sonicator, UV 

spectrophotometer, FTIR, stage 

micrometer microscope, scanning electron 

microscope, pH meter, brook field 

viscometer. 

Methods 

Preparation of microspheres by 

emulsion cross linking method 

Gelatine were accurately weighed and 

mixed in 10 ml of distilled water, 

preheated to 60ºc, followed by the addition 

of Tween 80 (0.1%w/v). To this, 1 g of 

amoxicillin trihydrate was added and 

thoroughly mixed to obtain a 

homogeneous solution. The mixture was 

maintained at 50ºc,and than added drop 

wise into 100 ml of liquid paraffin 

containing Span 80 (0.1 %w/v) preheated 

to 60 ºc at constant stirring with 3- blade 

stirrer in order to form w/o emulsion. 

Glutaraldehyde was added drop wise to the 

emulsion and stirring and stirred for 1 hr at 

room temperature to stabilize the 

microspheres. The mixture was then left to 

cool at between 5-10 ºc f 2or 30 min to 

enhance settling of the microspheres. 

Microspheres were collected by filtration 

using Whatman filter paper and washed 

with 3×10 ml of chloroform followed by 

2×10 ml of 5 %w/v sodium 

metabisulphite, dried at room temperature 

and transferred to glass vials [7] . 

Effect of process variables on 

microsphere properties. 

Gelatine microspheres were prepared at 

different stirring rates, cross linking agent 

(gluteraldehyde) amount, continues phase 

amount and with various drugs: polymer 

ratios as stated in Table 1 

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Table 1: Process variables 

S. N. Process variables Values 

1 Stirring rate (rpm) 500, 1000, 1500 

2 Cross linking agent amount (ml) 0.5, 1.0, 1.5 

3 Drug : polymer ratios 1:1, 1:2, 1:3, 1:4 

4 Continuous phase amount (ml) 100, 150, 200 

Working formula 

Table 2: Batches for selection of drug: polymer ratio 

Batch 

Drug : polymer 

ratio 

Glutaraldehyde 

amount (ml) 

Stirring rate 

(rpm) 

Continuous phase 

amount (ml) 

F1 1:1 0.5 500 100 

F2 1:2 0.5 500 100 

F3 1:3 0.5 500 100 

F4 1:4 0.5 500 100 

Table 3 : Batches for selection of glutaraldehyde amount and stirring rate 

Batch Drug: Polymer 

ratio 


amount (ml) 

Stirring 

Rate (rpm) 


amount(ml) 

F5 1:3 1.0 500 100 

F6 1:3 1.5 500 100 

F7 1:3 0.5 1000 100 

F8 1:3 1.0 1000 100 

F9 1:3 1.5 1000 100 

F10 1:3 0.5 1500 100 

F11 1:3 1.0 1500 100 

F12 1:3 1.5 1500 100 

Table 4: Batches to determine effect of continuous phase amount on microspheres 

Batch 

Drug: Polymer 

ratio 


amount (ml) 

Stirring 

Rate (rpm) 

Continuous Phase 

amount (ml) 

F13 1:3 1.0 1000 150 

F14 1:3 1.0 1000 200 


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Evaluation of microspheres 

Determination of percentage yield 

The prepared microspheres were collected 

and weighed. The measured weight was 

divided by total amount of all non-volatile 

components, which were used for the 

preparation of microspheres. The % yield 

was calculated following formula 

W Rec 

% yield = × 100 

Weight (drug + polymer) 

Where 

W Rec = Weight of microspheres recovered 

Determination of percentage of drug 

loading 

The percentage of Amoxicillin trihydrate 

loading in microspheres can be estimated 

using Eq1. 

L = 

Q m 

W m 

x 100 …(1) 

where, L is the loading (%) of 

microspheres, Qm is the quantity 

Amoxicillin trihydrate present in Wm of 

microspheres and Wm is the weight of the 

microspheres in grams. 

Determination of encapsulation 

efficiency 

The amount of Amoxicillin trihydrate 

encapsulated in the microspheres was 

determined using Eq 2. 

E = 

Q p 

Q t 

x 100 ….(2) 

Where E is encapsulation efficiency (%), 

Qp is the quantity of drug encapsulated in 

the microspheres (g), Qt is the actual 

quantity of drug used for encapsulation (g), 

and Qp is the product of drug content per 

gm of microspheres and yield of 

microspheres (g). 

Fourier Transform Infrared 

Spectroscopy 

Drug polymer interactions were studied by 

FTIR spectroscopy. The spectra were 

recorded for pure drug and drug loaded 

microspheres using FTIR. A pellet of 

approximately 1 mm diameter of drug was 

prepared by compressing 3-5 mg of the 

drug with 100-150 mg of potassium 

bromide in KBr press (Model M-15, 

Techno Search Instruments). The pellet was 

mounted in IR compartment and scanned 

between wave number 4000 – 400 cm-1 

using a Shimadzu Model 8400 FTIR. 

Particle size analysis 

The particle size analysis was carried out 

by using optical microscopy. The 

microspheres were analyzed for size and 

size distribution by first dispersing them in 

20 % v/v isopropyl alcohol to avoid 

swelling, vortexing for 3 min and 

ultrasonicating for 30 s before sampling. 

The particle size analysis was carried out 

by using optical microscopy. About 100 

microspheres were selected randomly and 

their size was determined by using optical 

microscope fitted with standard micrometer 

scale. 

Scanning electron microscopy 

The sample the scanning electron 

microscopy (SEM) analysis was prepared 

by sprinkling the microspheres on one side 

of the double adhesive stub. The stub was 

then coated with gold using Jeol JFC 1100 

sputter coater. The SEM analysis of the 

microspheres was carried out using Jeol 

JSM 5300, Japan. The microspheres were 


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viewed at an accelerating voltage of 15-20 

kV.heres in grams. 

Evaluation of in vitro release by static 

method 

Microspheres, equivalent to 10 mg of 

Amoxicillin trihydrate, were accurately 

weighed and transferred to 250 ml conical 

flask containing 100 ml phosphate buffer 

(pH 6.8). The flask was kept in an 

incubator at 37 °C, 1 ml samples withdrawn 

at regular intervals and, after suitable 

dilution, the amount of drug released was 

determined using a spectrophotometer at 

227.72 nm. Following each sample 

withdrawal, 1 ml of phosphate buffer was 

added to the release medium to replenish it. 

Five minutes before each sampling, the 

flasks were gently shaken by manually 

whirling it clockwise (15 revolutions) to 

minimize any concentration gradient within 

the release medium. The microspheres were 

allowed to settle down and clear 

supernatant medium withdrawn for drug 

analysis. The sample was filtered and the 

microspheres collected were transferred to 

the dissolution flask. Similarly, the release 

of Amoxicillin trihydrate from gelatin 

microspheres was determined 

spectrophotometrically at 227.72 nm 

(Shimadzu 1800) [5, 8] . 

Preparation of gel 

Preparation of the carbomer gel was based 

on a previously reported composition and 

method. Accurately weighed Amoxicillin 

trihydrate (100 mg) was added to 15 ml of 

water in a beaker and stirred well to 

dissolve the drug; 400 mg of carbopol was 

dissolved in this drug solution. Gelatin 

microspheres loaded with 100 mg of 

Amoxicillin trihydrate was added to the 

drug carbopol solution and mixed well. 


Methyl hydroxybenzoate (0.15 %w/w), 

propyl hydroxybenzoate (0.05 %w/w) and 

sodium metabisulphate (0.1 %w/w)) were 

then added to microsphere/drug/carbopol 

mixture, while stirring at 250 rpm, to obtain 

a homogenous mixture. Stirring was 

continued until a lump-free suspension was 

obtained. Thereafter, 0.33 ml of 

triethanolamine was added to produce a gel. 

This was followed by the addition of a 

sweetening agent (saccharin sodium, 0.1 

%w/w) and more water to make up to 20 g 

of gel [9] . 

Evaluation of bioadhesive gel 

incorporated microspheres 

Surface pH of the gel: 

An acidic or alkaline formulation is bound 

to cause irritation on mucosal membrane 

and hence this parameter assumes 

significance while developing a 

bioadhesive formulation. A digital glass 

electrode pH meter was used for this 

purpose. pH was noted by bringing the 

electrode near the surface of the 

formulations and allowing it to equilibrate 

for 1 min [10] . 

Viscosity Study: 

Viscosity of gels was studied on Brookfield 

viscometer by using spindle number 7 at 4 

revolutions per minute at constant 

temperature [11] . 

Estimation of drug content in formulated 

gels: 

Formulations containing 1 mg of drug was 

taken in 10 ml volumetric flask, dissolved 

in pH 6.8 phosphate buffer made up the 

volume to 10 ml with pH 6.8 phosphate 

buffer and then filtered. Absorbance values 

were measured at respective λmax (227.72 

nm) for drug. Concentrations of drug were 

calculated from the standard calibration 

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curve prepared in pH 6.8 phosphate buffers 

[12] . 

Bioadhesion study: 

In the present study, bovine cheek pouch 

was used as a model mucosal surface for 

bioadhesion testing. The bovine cheek 

pouch was procured from slaughter house, 

then excised and trimmed evenly from the 

sides. It was then washed in phosphate 

buffer (pH 6.8) and was preserved in the 

same or used immediately. The two sides of 

the balance were balanced with a 5 g 

weight on right hand side. The bovine 

cheek pouch excised and washed was tied 

tightly with the mucosal side upwards using 

a thread over the protrusion in the rubber 

block which is covered with inert 

aluminium surface. The block was then 

lowered into the glass container, which was 

then filled with isotonic phosphate buffer 

(pH 6.6) kept at 37°C±1°C, such that the 

buffer just reaches the surface of mucosal 

membrane and keeps it moist. This was 

then kept below the left hand set up of the 

balance. The film was then glued at the 

border adhered to a aluminium surface 

hanging on the left hand side and the beam 

raised, with the 5 g weight removed on the 

right pan side. This lowered the aluminium 

surface along with the film over the 

mucosa, with a weight of 5 g. The balance 

was kept in this position for 8 min and then 

slowly water was added to the plastic 

container in the right pan by pipette. The 

addition of water was stopped as soon as 

the detachment of two surfaces was 

obtained. Weight of water was measured. 

The excess weight in the pan i.e. total 

minus 5 mg is the force required to separate 

the film from the mucosa. This gave the 


bioadhesive strength of the formulation in 

grams [13] . 

Evaluation of in vitro release by dynamic 

method 

Evaluation in vitro release studies of 

Amoxicillin trihydrate from the carbomer 

gel was carried out at 37 °C using 

phosphate buffer (pH 6.8) as the release 

medium. A glass tube of 10 mm diameter 

and 100 mm height was taken. One end of 

the tube was closed using an egg membrane 

with the help of adhesive tape while the 

other end was kept open and used as drug 

reservoir compartment. Gel (1 g) 

containing Amoxicillin trihydrate was 

accurately weighed and transferred to the 

glass tube in a vertical position through the 

open end. The gel was gently pushed down 

to the surface of the egg membrane with the 

help of a stainless steel spatula to ensure 

that all the gel was in contact with the 

membrane. Phosphate buffer (2 ml, pH 6.8) 

was added to the reservoir compartment to 

wet the gel. The glass tube was placed in a 

beaker containing 100 ml of phosphate 

buffer (pH 6.8) such that that the egg 

membrane is just immersed in the 

phosphate buffer which acted as the 

receiving compartment. The receiving 

compartment was magnetically stirred (100 

rpm, Remi, India) at 37 °C. Samples (1 ml) 

were withdrawn from the receiving 

compartment at regular intervals and the 

amount of diclofenac sodium released from 

the gel was determined using a 

spectrophotometer at 227.72 nm 

(Shimadzu1800). After each withdrawal of 

sample, equal quantity of phosphate buffer 

was added to the receiving compartment to 

replenish it [14] . 

Release kinetics 

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Data obtained from in vitro release studies 

were fitted to various kinetic equations [15] 

to determine the mechanism of drug release 

from the microspheres. The kinetic models 

used were zero order equation, first order 

equation and Higuchi release using the 

following plots: Q t vs t, log (Q 0 -Q t ) vs t and 

Q t vs square root of t, respectively; where 

Q t is the amount of drug released at time t 

and Q 0 is the initial amount of drug present 

in the microspheres. T further, to ascertain 

the mechanism of drug release, the first 60 

% of drug release was fitted to Korsmeyer- 

Peppas 

model (Eq 3). 

Mt/Ma = kt n …. (3) 

Where Mt/Ma is the fraction of drug 

released at time, t, k is the rate constant and 

n is the release exponent. The n value is 

used to characterize different release 

mechanisms. 

RESULTS AND DISCUSSION 

Drug- excipients compatibility study 

FTIR spectrum of Amoxicillin trihydrate 

and drug loaded microspheres are shown in 

Figure 1 and 2 respectively. From the FTIR 

spectra of Amoxicillin trihydrate and drug 

loaded microspheres, it was found that drug 

and excipients are compatible with each 

other. 

Figure 1: FTIR spectra of Amoxicillin trihydrate 

Figure 2: FTIR spectra of physical mixture of Drug and excipients 


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Evaluation of microspheres for selection of drug: polymer ratio 

Table 5: Selection of drug: polymer ratio 

Batch Drug: Polymer Ratio %Drug Entrapment Particle Size(µm) % Yield 

F1 1:1 20±2.6 200±4.5 30±3 

F2 1:2 68±3.7 235±3.7 52±1.6 

F3 1:3 90±1.9 330±6.7 87±2.2 

F4 1:4 Lump formation - - 

‣ Drug: polymer ratio: As increase the 

polymer concentration, increase in 

particle size due to increase viscosity 

of emulsion medium is increased. Due 

to increase in viscosity, larger 

emulsion droplets which are difficult to 

break. But increasing the concentration 

of polymer, increase entrapment 

efficacy. Further increasing 

concentration of polymer, lump 

formation due to aggregates of 

microspheres. 

‣ In F3 batch highest drug entrapment 

obtained. So, Drug: Polymer ratio 1: 

3 was selected. For reducing particle 

size of microspheres stirring rate & 

glutaraldehyde 

amount optimized. 

Table 6: Selection of glutaraldehyde amount and stirring rate 

Batch 

Drug: Polymer 

ratio 


amount (ml) 

Stirring 

Rate (rpm) 

%Drug 

Entrapment 

Particle 

Size(µm) 

% Yield 

F5 1:3 1.0 500 92±2.4 155±3 85±2.4 

F6 1:3 1.5 500 91±4.8 170±1.9 90±1.9 

F7 1:3 0.5 1000 83±2.1 121±3.0 87±1.8 

F8 1:3 1.0 1000 94±3 80±0.9 94±2.6 

F9 1:3 1.5 1000 93±1.2 159±2 92±0.7 

F10 1:3 0.5 1500 

F11 1:3 1.0 1500 

F12 1:3 1.5 1500 

Fiber 

obtained 

Fiber 

obtained 

Fiber 

obtained 

Fiber 

obtained 

Fiber 

obtained 

Fiber 

obtained 

Fiber 

obtained 

Fiber 

obtained 

Fiber 

obtained 

‣ 

‣ Glutaraldehyde amount: increasing 

amount of cross linking agent retards the 

drug release. Crosslinking agent hardens 

the microspheres because of which drug 


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Cumulative % Drug Release 




release is decrease. Further increasing 

concentration, larger particle size due to 

adherence of excess cross linking agents 

on the surface of the microsphere. 

‣ Stirring rate: stirring speed was low 

(500 rpm), larger particle size obtained. 

Due to inadequate stirring speed which 

was not able to break the emulsion 

droplets. At higher speed, fiber 

obtained 15 . 

‣ From these results, F8 batch in which 

stirring speed 1000 rpm and 

glutaraldehyde amt is 1 ml.these 

microspheres had highest % drug 

entrapment and lowest avg particle size. 

So, F8 batch selected. 

Table 7: Effect of continuous phase amount on microspheres 

Batch 


amount (ml) 

Avg. particle size 

(µm) 

% Drug entrapment efficacy 

F13 150 160±3 62±3.6 

F14 200 155±1.9 57±2.7 

Values are means ± SD,( n=3). 

‣ Volume of continuous phase: Increase 

volume of continuous phase, emulsion 

droplets moved freely in medium, thus 

reducing collosion induced aggregates 

which could be the reason of high drug 

extraction in processing medium. 

Resulting in lower entrapment efficacy. 

Drug release study of microspheres (Batch F5 to F9) 

120 

100 

80 

60 

40 

20 

F5 

F6 

F7 

F8 

F9 

0 

0 100 200 300 400 

Fig 3: Cumulative Percentage drug released Vs Time 


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Cumulative % drug release 




In F8 batch 96.0% cumulative drug release 

obtained within 6 hrs so F8 batch is 

selected for incorporation in to gel. F8 

batch microspheres had smallest particle 

size and highest % drug entrapment. So, F8 

batch is selected. 

Scanning electron microscope 

Scanning electron microscopy (Fig 4) 

confirmed that prepared Microspheres were 

spherical in shape. Also average particle 

size of the prepared micro particles was 

within the size range of 50 to 90 µm. 

Table 8: Evaluation of bioadhesive gel 

incorporated amoxicillin trihydrate microspheres 

Evaluation 

Observation 

parameters 

Surface pH 6.9±0.1 

Viscosity 

4000±0.09 

(centipoises) 

Bioadhesive strength 6.50±0.100 

(g) 

Drug content (%) 99.25±0.03 

Fig 4: SEM photographs of gelatin 

microspheres loaded with Amoxicillin 

trihydrate 

IN-VITRO DIFFUSION STUDY 

‣ In-vitro diffusion study of bioadhesive gel incorporated Amoxicillin trihydrate loaded 

microspheres (Batch F8) and compared with microspheres (Batch F8). 

120 

100 

80 

60 

40 

20 

Amoxicillin trihydrate 

microspheres in gel 


microspheres 

0 

0 100 200 300 400 

Time (min) 

Fig 5: In-vitro release of Amoxicillin trihydrate from gelatine microspheres and 

microspheres-loaded gel. 


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The cumulative release of Amoxicillin 

trihydrate from microspheres and gel are 

shown in Figure 5. Drug from the 

microspheres was released in a controlled 

manner for ≥ 6 h. The release pattern was 

biphasic with an initial ‘burst’ release of 25 

% of the loaded drug was in the first 10 

min. Thereafter, release was slow but 

steady, and by the end of the 6 th hr, 96.1 % 

of the loaded drug was released. 

Amoxicillin trihydrate from the carbomer 

gel formulation is also shown in Figure 5. 

The release pattern was biphasic with an 

initial ‘burst’ release that was enhanced by 

the loading dose of Amoxicillin trihydrate. 

By the end of the first 10 min, 34 to 36 % 

of the drug was released; subsequently, 

release was teady and by the end of the 6 th 

hr, 95 % of the drug was released. 

RELEASE KINETICS 

The results of curve fitting into the 

mathematical models are given in Table 9. 

The results indicate the drug release 

behaviour from the formulated bioadhesive 

gel of Amoxicillin trihydrate. 

When the release rate of Amoxicillin 

trihydrate from the microsphere gel and 

their respective correlation coefficients 

were compared, it was found to follow 

Higuchi model kinetics(R 2 =0.938) 

indicating drug release was diffusion 

controlled. 

Table 9: Results of curve fitting of in vitro drug release from bioadhesive gel containing 

microspheres. 

Zero 

Formulation 

order 

First order Higuchi Korsmeyer pepps 

R 2 R 2 R 2 N 

Bioadhesive gel incorporated 


microspheres 

0.782 0.871 0.938 0.625 

For the tested formulation, the values of n 

on fitting the simple power equation 

Mt/M∞ = Kt n were found 0.625 for the 

release of Amoxicillin trihydrate from the 

bioadhesive gel, indicating anomalous (non 

fickian) diffusion, where drug release is 

controlled by combination of diffusion and 

polymer chain relaxation mechanisms. 

CONCLUSION 

The formulated Amoxicillin trihydrate 

loaded gelatine microspheres for sustain 

release was found to be potential and 

effective in terms yield, encapsulation 

efficacy, particle size and in-vitro release 

characteristics. The gel formulation , which 

consisted of drug loaded gelatine 

microspheres, showed sustain release of 

Amoxicillin trihydrate ≥ 6 hr, thus 

indicating their suitability for the sustained 

delivery of the drugs for the treatment of 

infections in periodontal pockets. However, 

further studies, including clinical tests are 

required to confirm the gel’s therapeutic 

efficacy. 

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