Development and Evaluation of Biodegradable Chitosan Microspheres Loaded with Ranitidine and Cross Linked with Glutaraldehyde
The present study aimed at the formulation of biodegradable chitosan microspheres loaded with ranitidine to overcome the poor bioavailability and frequent dose administration. Chitosan microsphere was prepared by simple emulsification technique by glutaraldehyde crosslinking. Various process variable and formulation variable such as speed of emulsification, cross linking time, drug/polymer ratio, volume of cross linking agent and volume of surfactant were optimized. Formulated microspheres were characterized for its entrapment efficiency, drug loading, in vitro drug release, Kinetics of drug release, surface morphology, particle size analysis, Fourier Transform Infrared Spectroscopy (FTIR) and Differential Scanning Calorimetry (DSC) thermal analysis. The characterized ranitidine microsphere formulations were investigated for in vivo gastric and duodenal antiulcer activity. The characterization of the fabricated microspheres showed smooth surface with narrow particle size distribution and entrapment efficiency upto 84%. The prepared microspheres exhibited a controlled drug release of 74% over a period of 24 h with initial burst release of 35% in the first 2 h. The FTIR and DSC reports showed that there was no potential drug interaction between the drug and polymer. In vivo studies shows that gastric volume, pH, total acidity and ulcer index of formulated ranitidine microspheres were significantly reduced as 2.67 mL, 5.59, 110 mEq L-1 and 1.74, respectively and also there is no evidence of extra tissue damage as seen in the biopsy report. From the data obtained it can be concluded that the chitosan microspheres could be considered as a potential biodegradable carrier for controlled drug delivery of ranitidine.
Received: September 25, 2010;
Accepted: October 21, 2010;
Published: March 29, 2011
Chitin is chemically (1→4)-2-acetamido-2-deoxy-β-D-glucan, i.e.,
abundant in nature and chitosan is its deacetylated derivative (Fig.
1), has many industrial applications such as lubricant, disintegrant, thickening,
stabilising and suspending agent in textile and paper industry (Upadrashta
et al., 1992), as a chelating agent for removal of harmful metals
in industrial nuclear wastes as a support for ion exchange, chelation affinity
chromatography (Qurashi et al., 1992). The industrial
source of chitin is shells of shrimp, lobster and crab. Chitin and Chitosan
are distinguished by their solubility profile.
|| Structure of chitosan
The characteristic properties of chitosan render them suitable for pharmaceutical
and biomedical application. Chitosan has antacid, antiulcer, hypocholesterolemic,
wound healing, haemostatic, spermicidal properties (Hillyard
et al., 1964). Chitosan has favourable biological properties like
biodegradability, biocompatibility and nontoxicity. Chitosan was found to improve
the fluidity of powder mixtures (Kumar, 2001). Chitosan
has good mucoadhesive property and show good application potential (Wittaya-Areekul
et al., 2006). Sustained release formulations have been successfully
prepared by using chitosan (Agnihotri and Aminabhavi, 2004;
Kofuji et al., 2005). Chitosan controlled drug
delivery systems for hormones (Berthold et al., 1996;
Cheng et al., 2005), vitamins (Shi
and Tan, 2002; Murata et al., 2002), proteins
(Remuman-Lopez et al., 1998; Grenha
et al., 2005) and enzymes (Chen and Chen, 1998;
Jiang et al., 1998) have been reported. The chitosan
has antitumour activity, thus chitosan microspheres bearing antineoplastic agents
are promising carriers for cancer treatment (Ouchi et
al., 1989). Chitosan also holds immense promise for ophthalmic delivery
(Paolicelli et al., 2009).
The pH dependant solubility of chitosan is a function of amino groups present
and is a drawback for oral delivery. Chitosan microspheres formed by electrostatic
interaction between a polyion and counterions become unstable in gastric fluid.
This problem can be countered by irreversible chemical cross linking (Berthold
et al., 1996). It is also eminent that the drug diffusion from a
chitosan microsphere could be effectively controlled by cross linking with a
dialdehyde such as glutaraldehyde (Thanoo et al.,
The drugs used in the treatment of ulcer include receptor blockers, proton
pump inhibitors, drugs affecting mucosal barrier and act on the central nervous
system (Manonmani et al., 1995). Even though wide
range of drugs available for the treatment of ulcer, many do not fulfill the
requirements and have many side effects such as arrhythmias, impotence and hemopoietic
changes are noted (Austin and Jegadeesan, 2003). H2
antagonists unlike anticholinergics they do not cause side effects like dry
mouth, urinary retention etc. They do not delay gastric emptying time which
may reflexly stimulate gastric secretion because of food remaining in the stomach
for long time. Also, it does not cause abdominal colic and diarrhea caused by
proton pump inhibitors (Goel and Shah, 2008). Out of the
available category of drugs for the treatment of ulcer, H2 antagonists
class of drugs like famotidine, ranitidine are considered to be the safest drugs.
Hence this drug has promising future if controlled release formulations are
Ranitidine is a H2 receptor antagonist. It is widely prescribed
in gastric ulcers, duodenal ulcers Zollinger-Ellison syndrome, systemic mastocytosis
(Aboofazeli and Shafaati, 2002) and gastroesophageal
reflux disease (Bruntan et al., 2006). H2
receptor antagonists not only inhibit gastric secretion, induced by histamine,
gastrin and cholinergic stimulation. They also promote healing of duodenal ulcers
(Sharma, 2007). The effects of factors such as food intake,
formulation, age and hepatic diseases, on blood concentrations of ranitidine
have been described by several researchers (Alkaysi et
al., 1989; Smith et al., 1984).
The H2 anti histamines like ranitidine is a very successful drug
due to its prominent clinical parameters. They block more than 90% of nocturnal
acid and 60-70% of day time secretion. The relative potency of ranitidine is
also higher when compared to other H2-antihistamines. The recommended
dose of ranitidine for duodenal, gastric ulcers, reflux esophagitis, NSAID ulcers
and Zollinger-Ellison syndrome is 150-300 mg BD (Reynolds,
Two types of polymorphic crystalline forms of ranitidine hydrochloride, Form
1 and Form 2 have been described. The basic form or Form 1 can be obtained from
the ethanolic solution of ranitidine base by salt formation with hydrochloric
acid. The filtration and drying characteristics of Form 1 are known to be unfavourable,
moreover, it exhibits considerable hygroscopicity. Form 2 is obtained upon the
isopropanolic recrystallization of Form 1. The preparation of Form 2 is described
in U.S. Pat. No. 4,672,133. The two above crystalline forms of ranitidine hydrochloride
are well distinguishable by X-ray powder diffraction patterns. From a technological
standpoint Form 2 is more advantageous, consists of larger crystals, is easy
to filter, to dry and less sensitive to moisture, Upon storage Form 1 slowly
gets converted into Form 2. The existence and spontaneous transformations of
polymorphic forms of drug substances are of disadvantage, because they cause
difficulties to fulfill exacting pharmaceutical requirements and specifications.
The physicochemical properties of products with such polymorphics change according
to the actual ratios of polymorphic forms. The sharp endothermic heat low peak
characteristic for the melting of Form 2 ranitidine hydrochloride is seen at
143-145°C and this is the same on the DSC curve of the mechanical mixture
(Wilfried and Karin, 1997).
It has been reported that the oral treatment of gastric disorders with H2
antagonist like ranitidine or famotidine used in combination with antacids promotes
local delivery of these drugs to the receptor of parietal cell wall. Local delivery
also increases the stomach wall receptor site bioavailability and increases
efficacy of drugs to reduce acid secretion. Hence this principle may be applied
for improving systemic as well as local delivery of ranitidine, which would
efficiently reduce the gastric acid secretion (Coffin and
From the above facts, a need was felt to develop a preparation that deliver ranitidine in the stomach and would increase the efficiency of the drug, providing sustained action. Thus an attempt was made to prepare ranitidine loaded chitosan biodegradable microspheres.
MATERIALS AND METHODS
Materials: Chitosan (medium mol wt. ca 40 kDa) was obtained from Central Institute of Fisheries and Technology, Cochin, India. Ranitidine was obtained as a gift sample from Novartis Bombay. Sorbitan sesquioleate, glutaraldehyde (25% aqueous), liquid paraffin light with viscosity of 18 CPS, petroleum ether were obtained from Loba Chemie Pvt. Ltd., Bombay. The entire research project was carried out in Research Lab., GIET School of Pharmacy, Rajahmundry, India during 15 June 2007 to 14 Sep., 2010.
Preparation of Ranitidine loaded chitosan microspheres: Famotidine containing
chitosan microspheres were prepared by simple emulsion technique. In the preliminary
preparations various ratios of drug polymer were tried. Four percent solution
of chitosan in 5% aqueous acetic acid containing 2% NaCl was prepared and the
drug was loaded by mixing the required amount of drug with 6 g of chitosan paste
and it was dispersed in a mixture of 35 mL liquid paraffin and 25 mL of petroleum
ether containing 0.85 g of sorbiton sesquioleate in a 100 mL round bottomed
flask at room temperature (Jameela and Jayakrishnan, 1995).
The dispersion was stirred using stainless steel half moon shaped paddle stirrer
at 2000 rpm for 5 min and 10 mL of Glutaraldehyde Saturated Toluene (GST) prepared
according to the Patel method (Patel and Patel, 2007),
was introduced into the flask while stirring. At the end of 30 min, glutaraldehyde
(25% v/v aqueous solution) was added and stirring was continued. The volume
of cross linking agent and cross linking time was varied in preliminary trial
batches from 0.5-15 mL and 1-3 h, respectively. The stirrer speed was also varied
from 1500-3000 rpm. The stirring was continued for a total duration of 90 min,
at the end the hardened microspheres were filtered, washed several times with
petroleum ether followed by acetone, a 5% solution of sodium metabisulphate
and finally with water. The microspheres thus obtained were dried overnight
in an air oven at 60°C. The microspheres were stored in a dessicator.
Determination of loading efficiency: Ranitidine content in the preparation
was determined by extracting the drug containing microspheres using pH 6.8 phosphate
buffer. Fifty milligram of microspheres were taken and triturated and dissolved
in 50 mL of pH 6.8 phosphate buffer. The solution was filtered through Millipore
filters and the amount of drug was measured after suitable dilution at 226 nm
by spectrophotometry. The amount of drug loaded in microspheres was calculated
by the following formula (Gladiziwa and Klotz, 1993).
||Weight of microspheres in grams
|| Quantity of drug present in Wm g of microspheres
Entrapment efficiency: Fifty milligrams of accurately weighed microspheres
were crushed in a glass mortar-pestle and the powdered microspheres were suspended
in 10 mL of pH 6.8 phosphate buffer solution. After 24 h the solution was filtered
and the filtrate was analysed for drug content. The drug entrapment was calculated
using the formula (Patel and Patel, 2007).
||Weight of drug present in microspheres (practical drug content)
||Theoretical weight of drug
Particle size analysis: The size distribution in terms of average diameter
(davg) of microspheres was determined using the optical microscopic
method. Scanning Election Microscopy (SEM) was performed to characterize the
surface morphology of formed microspheres (Thanoo et
al., 1993) by using Hitachi S-520 SEM.
Fourier transform infrared spectroscopy: The FT-IR spectrum of chitosan, ranitidine and ranitidine loaded chitosan microspheres were recorded on a PerkinElmer (model No-Spectrum Rx, Serial No. 83806) instrument using KBr discs in the range of 4000-400 cm-1. FT-IR spectrum could be a useful tool in determination of structural change undergone by the drug or the polymer in the microsphere formulation.
Differential scanning calorimetry: Differential Scanning Calorimetry (DSC) of ranitidine, blank microspheres and drug loaded microspheres were performed with DSC 821e Mettler Toledo. The DSC tracings were performed from 20 to 240°C at a rate of 10°C min-1.
In vitro release study: In vitro release study was carried
out in pH 6.8 phosphate buffer solution at 37±1°C. Drug loaded microspheres
(50 mg) were added to 50 mL of dissolution medium in a stoppered bottle. The
bottle was fixed in the orbital shaker (Remi) and shaker speed was adjusted
to 50 rpm. The samples were collected at predetermined time intervals (30 min,
1, 2, 4, 6, 8, 10, 12, 16, 20, 24, 36 and 48 h) for analysis. The medium was
replenished with an equal volume of phosphate buffer solution after withdrawal
of each sample. Values reported are the average of three determinations using
the same technique (Jaimini et al., 2007).
Kinetics of drug release: In order to understand the mechanism and kinetics
of drug release, the result of the in vitro dissolution study of microspheres
were fitted with various kinetic equations, such as zero-order (percentage release
versus time), first-order (log percentage of cumulative drug remaining versus
time), Higuchis model (percentage drug release versus square root of time)
(Tamizharasi et al., 2008).
In vivo gastric and duodenal antiulcer activity
Pyloric ligation: Wistar albino rats of both sex were grouped into
eight each containing 6 animals. They were kept in the animal house at room
temperature 25+2°C, with relative humidity of 45-55% maintained under 12
h light and dark cycle and were fed with standard rat feed and were acclimatized
for a week before the study (Bhave et al., 2006;
Kath and Gupta, 2006). Group I served as normal control
in which distilled water was administered orally in which no pyloric ligation
was done, group II served as disease control, group III received Ranitidine
50 mg kg-1 orally and it was considered as standard, group IV served
as Ranitidine Formulation group and the dose equivalent to ranitidine 50 mg
kg-1 was administered.
Rats were fasted for 36 h prior to the surgical procedure and kept in raised mesh-bottomed cages to avoid coprophagy. Under ether anesthesia the abdomen was opened by a small midline incision below the xiphoid process. The pyloric portion of the stomach was identified, slightly lifted, avoiding traction to the pylorus or damage to the blood supply. The stomach was then replaced carefully and the abdominal wall closed by interrupted sutures. Animals were deprived of both food and water during the post operative period and were sacrificed at the end of 19-20 h after the operation. The stomach was dissected out as a whole by passing a ligature at the esophageal end.
The stomach was separated from the surrounding tissues and organs and thus
brought out as a whole along with its contents. The contents were subjected
to centrifugation (3000 rpm for 10 min) and then analyzed for mean volume of
gastric secretion, mean pH and mean total acid. The pH was estimated by using
indikrom pH strips (Glaxo India Limited, India) with pH ranges of 2-4.5 and
5-8.5 with a difference range of 0.5. Free acidity and total acidity were estimated
by titrating 1 mL of centrifuged sample with 0.01 N NaOH, using Topfers reagent
as indicator and phenolphthalein indicator respectively. Acidity was expressed
in clinical units that are the amount of 0.01 N NaOH base required to titrate
100 mL of gastric secretion (Kulkarni, 1985).
Acidity was expressed as:
Aspirin induced ulcer: In Aspirin induced ulcer models four groups of
albino rats of either sex weighing 150-175 g, with each group consisting of
six animals were used. The first group served as a normal control the second
group served as disease control and the third group served as standard group
that received ranitidine 50 mg kg-1 and group four received Ranitidine
formulation equivalent to ranitidine 50 mg kg-1. All the animals
received above treatment once daily for eight days orally. After 8 days of treatment,
animals were fasted for 24 h. Ulcer was produced by administration of aqueous
suspension of aspirin (200 mg kg-1 orally) on the day of sacrifice.
The animals were sacrificed 4 h later and stomach was opened to calculate the
ulcer index by kunchandy method (Kunchandy et al.,
The antiulcer activity was carried out after the ethical approval from CPCSEA and it was done as per the recommended guidelines of CPCSEA reg. No. 1069/AC/07/CPCSEA.
Statistical analysis: Statistical analysis was performed using graph pad Instat 3 software. All the tests were run in triplicate (n=3). Experimental results were expressed as Mean±SD and one way ANOVA for significance at p<0.05 was conducted for the release profiles.
Ranitidine loaded chitosan microspheres were prepared by simple emulsification
phase separation technique. Chitosan was selected as a polymer for the preparation
of microspheres owing to its biodegradable, antiulcer, mucoadhesive properties
and it may give better synergistic effect for the treatment of ulcer. Different
concentrations of acetic acid from 1 to 6% w/v were used for preparing polymer
solution, but 5% w/v acetic acid was finally used owing to its good solubility
of chitosan and maximum sphericity was observed. Therefore from the preliminary
trials it was decided that 4% chitosan solution and 5% acetic acid was found
to be optimum concentration for the polymer solution. Liquid paraffin was used
as the dispersion medium. 0.85 mg of sorbiton sesquioleate was added to dispersion
medium and it was found to minimize aggregation of microspheres (Patel
and Patel, 2007).
Preliminary trial batches were prepared to study the effect of the volume of cross-linking agent (glutaraldehyde), time of cross linking and stirring speed, drug entrapment efficiency and characteristics of the microspheres. The volume of glutaraldehyde saturated in toluene was varied from 0.25 to 15 mL. Discrete spherical spheres were obtained using 15 mL of glutaraldehyde saturated in toluene. Batches prepared using 0.25-5 mL of GST yielded irregular microspheres. The higher amount of glutaraldehyde appears to favour the cross linking reaction and spherical free flowing microspheres were obtained using 10 mL of GST. The entrapment efficiency was also good and it was found to be 84%. Thus, we can conclude that 10 mL GST was the optimum volume for the preparation. Increase in cross linking time (1-3 h) in all preliminary trial batches affected the release rate. The cross linking polymer and time probably becomes more rigid and thus decrease the release of drug. The cross linking time did not have significant effect on percentage drug entrapment efficiency.
The chitosan ranitidine microspheres made with 1:2 drug polymer ratio at 2000 rpm stirring speed with 10 mL GST as cross linking agent had higher loading efficiency of ranitidine and further increase in drug polymer ratio does not give increase in loading (Table 1).
Analysis of particle size showed that 35% were below 75 um, while 40% had particles
sizes from 75-150 um and 25% were in the range from 150-300 um. Extensive analysis
on particle size gave us the information that lower stirring speed of 2000 rpm
has produced higher percentage of larger spheres ranging between 150-300 μm.
|| Preparation and characteristics of chitosan microspheres
(n = 3)
||Scanning electron micrographs of (a, b) Batch R2 Chitosan
ranitidine microspheres and (c, d) batch R3 Chitosan ranitidine microspheres
The release rate was extended upto 24 h for the batch having larger spheres.
Drug release was found to be more faster from the spheres of smaller size due
to the increased surface area in contact with the dissolution medium (Jameela
and Jayakrishna, 1995).
The microspheres obtained were fairly spherical in shape, as evidenced by scanning electron microscope analysis (Fig. 2). The morphology of the drug loaded microspheres appeared to be little rough.
UV spectrophotometric method was employed to determine the encapsulation efficiency
of ranitidine in the microsphere prepared. The entrapment efficiency was determined
by measuring the absorbance at 226 nm using pH 6.8 phosphate buffer solution.
||FTIR spectras of (A) Chitosan microspheres, (B) Pure ranitidine,
(C) Ranitidine loaded chitosan microspheres
The maximum percentage of encapsulation was found to be 84% for the batch R2
cross linked with 10 mL of GST at slow stirring. Many factors affect the entrapment
efficiency of drugs in chitosan microspheres, e.g., nature of drug, chitosan
concentration, drug polymer ratio, stirring speed etc. Generally lower concentrations
of chitosan shows low entrapment efficiency (Orienti et
al., 1996), however at higher concentrations chitosan forms highly viscous
solution which are highly difficult to process.
The FTIR spectra of chitosan depict characteristic absorption band at 3437 cm-1 which represents the presence of hydrogen bonded OH group. The amino group has a characteristic absorption band in the region of 3400-3500 cm-1 which must have been masked by the absorption band due to O-H group. Chitosan showed the characteristic bands of the amide at 1654, 1608 and 1323 cm-1. The ether linkage has a characteristic band at 1091 cm-1. The FTIR spectra of ranitidine showed characteristic absorption band at 3435 cm-1 which represents the presence of N-H str of secondary amines. The weak absorption band at 3050 cm-1 represents the presence of furan ring. Ranitidine showed the characteristic Nitro band at 1357 cm-1, The absorption band at 650 cm-1 represents L-S Str.
FTIR spectra of ranitidine loaded microspheres showed absorption bands at 283, 1458 and 1367 cm-1 of glutaraldehyde crosslinked chitosan microspheres are rather intense as a consequence of enhanced aliphatic C-H str absorption. The shift of the sharp peak from 1600 to 1620 cm-1 stands for the stretching vibrations of C=N in shiffs base formed by the reaction of glutaraldehyde and chitosan. The presence of N-H band at 3402 cm-1, C-H band at 650 cm-1 proves that there is no change of functional groups present in ranitidine. The FTIR spectra of chitosan, ranitidine and ranitidine loaded chitosan microspheres are shown in Fig. 3.
As DSC is a useful tool to monitor the effects of additives on the thermal
behavior of materials, this technique was used to deliver qualitative information
about the physicochemical status of drug in the preparations (Dhanikula
and Panchagnula, 2004). Thermogram of chitosan showed a broad peak at 58°C
over a large temperature range is attributed to water loss due to evaporation
of absorbed water and this represents the energy required to vapourise water
present in the samples. DSC thermograms of chitosan, ranitidine and ranitidine
loaded chitosan microspheres are shown in Fig. 4. Under the
experimental conditions no degradation DSC peak was observed for chitosan polymer
that normally occurs at 280°C (Cervera et al.,
2004; Liao and Hung, 2004).
||A: DSC of chitosan, B: DSC of pure ranitidine and C: DSC of
ranitidine loaded chitosan microsphere
||Release profiles of ranitidine microspheres batches-R1, R2,
R3, R4 and R5
DSC thermogram of ranitidine form II (Fig. 4) shows sharp
endothermic peak at 144°C which corresponds to their melting point temperature
range of 143-145°C. The peak disappeared for the drug loaded chitosan microspheres,
which indicated that the drug was molecularly dispersed inside of the matrix
of chitosan as a solid solution.
The ranitidine release profiles from chitosan ranitidine microspheres at 37°C
pH 6.8 were shown in Fig. 5. The drug release from the microsphere
formulations were found to be 52, 74, 71, 58 and 54% at the end of 24 h for
the batches R1, R2, R3, R4 and R5, respectively. From the release profile, it
is clear that the release of ranitidine from the microsphere was characterized
by initial rapid release (burst release) phase of 35% which is related to the
drug entrapped near the surface of the microsphere, followed by a slow release
phase. The drug release was found to be rapid in batch R2 due to its smaller
particle size and it also exhibited initial rapid burst release. The release
was found to be highest at 24 h and 74% release was found. This was exhibited
by 1:2 drug polymer ratio batch. 1:3 batch did not show much variation. The
remaining batches exhibited less drug release. As the cross linking agent and
time increases the release rate is extended, this may be due to hardening of
the microspheres as the time increases.
|| First order release kinetics
|| Zero order drug release
|| Higuchi release kinetics
Ten milliliter cross linking agent (GST) and 3 h cross linking period exhibited
formation of good microspheres with 74% release within 24 h.
The in vitro release data were applied to various kinetics models to
predict the drug release mechanism and kinetics (Fig. 6-8).
The drug release mechanism from the microspheres thus can be described as diffusion
controlled. The drug release was proportional to square root of time. When percentage
of cumulative drug released versus time was plotted in accordance with first-order
and zero-order equations, the r2 values obtained were found to be
better for first-order plot compared to zero-order drug release, indicating
that the drug release was described better with first-order release kinetics.
|| Antiulcer effect of ranitidine microsphere formulation on
pyloric ligation induced gastric ulcer in rats
|Values are expressed as mean ±SEM, n=6 in each group.
||Biopsy of Rat Stomach induced with ulcer. (a) Section of stomach
from normal control rat shows normal architecture, (b) Section of stomach
from disease control rat shows severely damaged cells, (c) Section of stomach
from Ranitidine treated rat shows mild damaged cells and (d) Section of
stomach from Ranitidine microsphere formulation treated rat shows mild damaged
In aspirin and pylorus ligation induced gastric ulcer models the microsphere
formulation reduced the gastric volume, total acidity and ulcer index (Table
2) thus showing the anti secretory mechanism involved in the antiulcerogenic
activity through H2 receptors. Ulcer index parameter (Table
3) was used for the evaluation of antiulcer activity since ulcer formation
is directly related to the factors such as gastric volume and total acidity
(Goel and Bhattacharya, 1991). From the results it is
clear that gastric volume, pH, total acidity and ulcer index of formulated ranitidine
microspheres were significantly reduced as 2.67 mL, 5.59, 110 mEq L-1
and 1.74, respectively. The biopsy reports of all the groups of rats were analyzed
and shown in Fig. 9a-d.
|| Antiulcer effect of Ranitidine microsphere formulation on
aspirin induced gastric ulcer in rats
|Values are expressed as Mean±SEM, n = 6 in each group.
Chitosan was selected as a polymer for the preparation of microspheres owing to its biodegradable, antiulcer, mucoadhesive properties and it may give better synergistic effect for the treatment of ulcer.
The production of ranitidine microspheres is based on the solubility behavior of chitosan, which is poorly soluble in water. Addition of an acid improves the solubility as a result of the protonation of the amino groups. The solubility is also dependent on other anions present in the solution. The presence of acetate, lactate or glutamate, chitosan shows good solubility. Where as phosphate, polyphosphate, sulphate and glutaraldehyde decreases the solubility. For this reason acetic acid was selected to dissolve the chitosan and glutaraldehyde was used for microsphere formation. Glutaraldehyde leads to a poorly soluble chitosan derivative where by microsphere formulation become possible.
The microspheres obtained were fairly spherical in shape, using 15 mL of glutaraldehyde
saturated in toluene and similar results were obtained by Guerrero
et al. (2010). When the volume of glutaraldehyde used in the microsphere
preparation was 15 mL, the cumulative percentage of drug released was lowered.
This is because increase in glutaraldehyde concentration caused highly crosslinked
spheres and become dense.
Jameela and Jayakrishnan (1995) described, during the
cross-linking and hardening process, water is exuded from the microspheres along
with the dissolved drug and this appears to be responsible for the rather low
incorporation efficiency. Increase in stirring speed beyond 3000 rpm has decreased
the particle size upto 10 μm. In this case the drug release was found faster
due to its increased surface area in contact with the dissolution medium. Increase
in the cross linking time favoured the controlled release of drug from the spheres.
This is also due to the hardening of the spheres with longer cross linking time.
The high entrapment efficiency was similar to that reported for the encapsulation
of drugs that were soluble in the same solvent as polymers using the spray drying
technique (Blanco et al., 2003). The drug content
was increased upto the drug polymer ratio 1:3 and the entrapment efficiency
was increased upto the drug polymer ratio 1:4. But at higher drug polymer concentration
both the drug content and entrapment efficiency was decreased. At higher polymer
concentrations viscosity of chitosan became too high, as a consequence a homogeneous
distribution of the added glutaraldehyde was not possible, which leads to the
formation of larger particles with reduced drug content and entrapment efficiency.
Exactly the insufficient amount of crosslinking agent for higher polymer concentration
and inadequate homogeneous dispersion cause slower crosslinking and insufficient
entrapment ability that leads to decrease in drug content and entrapment efficiency.
Chitosan microparticles prepared by Huang et al.
(2003) using a spray-drier showed a particle size of 2.12 μm and their
external surface appeared smooth. In our study the average particle size was
increased with increase in drug polymer ratio. When the polymer amount was increased
more viscous internal phase occurred during the emulsification process, the
internal phase was hardly dispersed in the outer phase and larger micropsheres
were produced, which lead to increase in average particle size.
The in vitro release profile of R1, R2 and R3 mainly depends on entrapment efficiency. Even though the drug polymer ratio was increased upto 1:3 in formulation R3, it showed higher cumulative percentage of drug release due to its higher entrapment efficiency. Thereby the increase in drug release was in the order of R1< R2< R3. Incase of formulation R4 even though having higher entrapment efficiency than R3, it showed lesser drug release due to its higher polymer concentration. Batch R5 had both lower entrapment efficiency and higher polymer concentration which lead to a marked reduction in the drug release.
From the in vitro release profiles of various ranitidine loaded microspheres,
the formulation R3 which contains 1:3 drug polymer ratios could be produced
successfully with rapid burst release of 26.93% which is related to the drug
entrapped near the surface of the microsphere, followed by subsequent slow sustained
release of 85.61% upto 24 h. This burst phase has been also described for the
release of different drugs from chitosan microspheres (Blanco
et al., 2000; Corrigan et al., 2006;
Huang et al., 2003). The burst effect observed
in drug release from micro particulate system is not itself an advantage or
disadvantage of the formulation; it depends on the type of drug entrapped and
also on the type of application of the microspheres (Trapani
et al., 2003). Further Higuchi release kinetics was also achieved.
So, the formulation R3 will be considered as best formulation to overcome the
frequent administration of ranitidine tablets and to improve the patient compliance.
It was also confirmed that there was no potential drug interaction produced
between polymer, drug and other ingredients as evidenced from the FTIR and DSC
The biopsy reports of all the groups of rats were analyzed and shown in Fig.
9a-d and it was found that the section of stomach from
normal control rat showed normal architecture, section of stomach from disease
control rat showed severely damaged stomach cells with chronic inflammation,
section of stomach from Ranitidine treated rat showed mild damaged cells and
the section of microsphere formulation treated also showed mild damaged cells
confirming the antiulcer effect of formulated Ranitidine microspheres and also
there is no evidence of extra tissue damage as seen in the biopsy report.
Ranitidine loaded chitosan microspheres using glutaraldehyde as crosslinking agent by simple emulsion technique could be produced successfully with rapid burst release of 35% and subsequent slow sustained release of 74% upto 24 h. Chitosan being natural biodegradable polymer gives no toxicity when incorporated in formulations. The present ranitidine loaded microspheres are suggested to be useful for the improvement of ranitidine efficacy against peptic ulcers.
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