ISSN: 0975-8585
October – December 2011 RJPBCS Volume 2 Issue 4 Page No. 662
Research Journal of Pharmaceutical, Biological and Chemical
Sciences
p
H
-Responsive and Mucoadherent Microparticles for Oral Delivery of
Serratiopeptidase: Development and In Vitro Characterization
Preeti K Suresh1
* and Anish Chandy2
1 University Institute of Pharmacy, Pt. Ravishankar Shukla University, Raipur (CG) INDIA
2 School of Pharmacy, Chouksey Engineering College, Bilaspur (CG) INDIA
ABSTRACT
The aim of the present study was to explore the potential of enteric coated chitosan microspheres as
carriers for oral site-specific delivery of peptides and proteins. In this study, microspheres of serratiopeptidase
were prepared by water in oil (w/o) emulsification solvent evaporation technique. Ratio of chitosan and
serratiopeptidase was varied in the range of 1:1, 1:2 and 2:1. The microspheres were then coated with Eudragit L
100. The product was obtained as non-aggregated free flowing microparticles. The drug content was found to be
between 15 to 40%. The presence of protein drug in the microparticles was confirmed by SDS-PAGE technique. In
vitro drug release studies indicated pH-responsive release profile. In vitro release of the drug first into phosphate
buffer pH 1.2 for 1 hr and then in phosphate buffer pH 6.8 at 37ºC was determined as a function of crosslinking
coating density of the microparticle. The pH-responsive behavior of the microparticle was confirmed, as there was
less than 10% release in phosphate buffer pH1.2. The extent of drug release had a remarkable dependence on the
coating density of the Eudragit L 100, the highly crosslinked spheres releasing only around 35% of the incorporated
drug in 6 hour compared to 40% from lightly coated spheres. The mucoadhesive nature of the microparticles was
established by an in situ method. These results indicate that Eudragit L 100 coated chitosan microparticles are
promising carriers for pH-responsive oral drug delivery of peptides and proteins.
Keywords: peptide, Eudragit L 100, Chitosan, pH-dependent, pH-sensitive, intestine-specific.
*Corresponding author
Email: preeti_venugopalan@yahoo.co.inINTRODUCTION
The intestinal drug targeting has been exploited for systemic delivery of active drugs. Most of the
peptide and protein drugs are unstable in the stomach and upper part of intestine. Apart from stability
problems, peptides are not well absorbed from the lumen of the GIT due to their large molecular size and
have high sensitivity to brush border peptidase activity. Comparatively, proteolytic activity of intestinal
mucosa is less than that observed in the stomach. Intestinal specific drug delivery systems protect peptide
drugs from hydrolysis and enzymatic degradation in the stomach, and eventually release drugs in the
jejunum, deuodenum, ileum or colon, which promises greater systemic bioavailability.
Serratiopeptidase (STP) is an anti-inflammatory, proteolytic enzyme isolated from the
microorganism, Serratia sp. HY-6 and is orally active [3]. Oral proteolytic enzymes have been
used successfully for inflammatory conditions. Recently, the intestinal absorption of orally
administered STP has also been demonstrated. To achieve an ideal therapeutic effect, however,
it is essential that any enzyme preparation be enterically coated so as to release the enzymes in
the intestines (where they can be absorbed) and not in the stomach (where they can be
digested). STP degrades in the stomach, but it is well absorbed from the small intestine.
Microsphere based drug delivery systems have received considerable attention in
recent years. The most important characteristic of microspheres is the microphase separation
morphology, which endows it with a controllable variability in degradation rate and drug
release.
pH-activated drug delivery systems permit the drug release at specific pH and the drug
molecules can thus be protected from degradation in the hostile gastric environment and in the
intestinal fluid the polymers dissolve and release the drug at intestinal pH>6.8. These types of
systems can be fabricated with polymers which are insoluble in acidic pH of stomach, but
soluble in basic pH prevailing in the intestine, viz., Eudragit L100, Eudragit S, Eudragit L, HMCP,
etc.
The aim of the present of work was to achieve site-specific controlled delivery of STP in
the small intestine. To achieve this objective Eudragit coated chitosan microparticles of STP were
prepared. The Eudragit coating will dissolve in the small intestine leaving chitosan microsphere,
which swells in the intestinal fluid leading to controlled release of STP from the microspheres.
The proposed mechanism is illustrated in Figure 1.
Fig. 1: Proposed mechanism of Eudragit®
coated chitosan microsphereshttp://www.rjpbcs.com/pdf/2011_2(4)/[70].pdf
ISSN: 0975-8585
October – December 2011 RJPBCS Volume 2 Issue 4 Page No. 664
MATERIAL AND METHODS
Materials
Chitosan (degree of deacetylation, 85.2%) particles with a viscosity 50 cps was obtained
as a gift sample from CFTI, Kochi, India. Serratiopeptidase was obtained from Centaur Labs Ltd,
Mumbai, India, as a gift sample. All other reagents used were of analytical grade from different
commercial manufacturers.
Development of Microparticles
Preparation of STP-containing chitosan microspheres (Chi-STP)
Chitosan (Chi) microspheres containing STP were prepared using w/o emulsification–
solvent evaporation method as reported by Hori et al [2]. Chitosan (100 mg) was dissolved in 5
ml of a 1% (v/v) acetic acid aqueous solution, and to this 2 ml of aqueous solution containing
STP (100 mg) was added. The chitosan (Chi) solution containing STP was dripped over 5 min into
200 ml of liquid paraffin containing SO-15
®
at 1% (w/v), which was continuously stirred at 500
rpm. The w/o emulsion obtained was stirred vigorously at 35C for 24 h, diethyl ether was
added, and the mixture was further, stirred overnight. The particles obtained were washed with
diethyl ether, 25% (w/w) ammonia aqueous solution, ethanol and diethyl ether in that order.
The particles were obtained by filtration and dried in air to yield microspheres.
ER-coated Chi-STP microspheres
Eudragit®
(ER)-coated Chi-STP microspheres (ER-Ch-STP) were prepared by coating Chi-
STP with Eudragit L 100 following the method reported by Bogataj et al [1]. Eudragit L 100 was
dissolved in 20 ml of acetone and the chitosan microspheres were dispersed in it. Then
equivalent amount of magnesium stearate was added and the dispersion was added dropwise
to 100 ml liquid paraffin (Heavy: Light = 1:1). To this dispersion 20 ml hexane was added with
stirring. The mixture was stirred for 90 min and the microspheres formed were collected by
filtration and washed with hexane twice and dried at room temperature.
Optimization of Process Variables
Various process variables, which could affect the preparation and properties of the
microspheres, were identified and studied. Preparation of pH-responsive microspheres involves
various process variables out of which drug: polymer ratio (D), temperature (T) and coating
polymer: microsphere ratio (P) was selected for the optimization of formulation.
The formulation code and respective variables used in the preparation of microspheres
are listed in Table 1. The effect of these variables was observed on particle size and size
distribution, % drug entrapment and % yield of microspheres.
CHARACTERIZATION
Size distribution and morphology
Scanning Electron Microscopy (SEM JEOL JSM-5800, Japan) was used to evaluate the
surface texture, shape and size of the microspheres. The samples for SEM were prepared by
lightly sprinkling the microspheres powder on a double adhesive tape, which stuck to an
aluminum stub. The stubs were then coated with gold to a thickness of about 300A using a
sputter container. The photomicrographs are shown in Figures 2 and 3.
Yield
To determine the % yield, weight of prepared microspheres was divided by the total
weight of all the non-volatile components used for the preparation of the microspheres. The
results are presented in Table 2.
Drug content
Chi-STP microspheres (10 mg) were dissolved in 10 ml of SIF (pH 7.4) by stirring with a
vortex mixer. An aliquot of 150 μl of the solution was treated with casein solution to detect itsproteolytic activity and to measure the amount of STP from the standard curve. The resultant
solution was centrifuged at 3000 rpm for 5 min and the supernatant was analyzed
spectrophotometrically at 280 nm.
Table 2: Encapsulation efficiency, yieldIn vitro release profile
Accurately weighed (60 mg) microspheres equivalent to 10 mg drug were suspended in
500 ml of mixed phosphate buffered saline (pH 6.8), and incubated at 37o
C and 90 rpm. After 1,
2, 3, 4, 5, 6 and 24 h incubation, 1ml of medium was collected, 4.5 ml Casein solution added
and allowed to stand for 37o
C for 20 min. [5] To the above solution, and trichloroacetic acid was
added to break the reaction. The supernatant collected and was used for the quantitative assay
of STP. For this assay, the final sample was obtained after centrifugation at 10,000 rpm for 5
min, and absorbance was measured spectrophotometrically at 280
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ER-Chi-STP accurately weighed microspheres equivalent to 10mg drug were suspended
in 500 ml of phosphate buffered saline (pH 1.2) and incubated at 37o
C at 90 rpm. The samples
were collected at regular intervals of 15 min. The pH was then adjusted to pH 6.8. At 1, 2, 3, 4,
5, 6 and 24 h after the start of the incubation in mixed phosphate buffered saline (pH 6.8), 1ml
of the medium was taken, 4.5 ml casein solution was added and allowed to stand at 37o
C for 20
min. Trichloroacetic acid was added to this solution to break the reaction and then centrifuged
at 10000 rpm for 10 min. The supernatant was used for the measurement of the amount of STP
released. Proteolytic activity was determined to measure the concentration of STP by
spectrophotometric estimation at 280 nm. The results are presented in Figures 4-6.
Fig. 4: Drug release profile of various microparticles (drug: polymer ratio of 1:1)
Fig. 5: Drug release profile of various microparticles (drug: polymer ratio of 1:2)
Detection of Drug by SDS PAGE
10 mg microspheres were dissolved in 1ml phosphate buffered saline (pH 7.4), then
vortexed for 10 min, allowed to stand for 5 min and again vortexed for 5 min to obtain
equilibrium concentration of drug in solution. The sample was centrifuged at 10,000 rpm for 10
min. Supernatant was collected and used for SDS- PAGE. 20µl of supernatant was taken and
applied on gel with reducing buffer and dye methylene blue. The stain was detected by marker
silver stain. This procedure was repeated for non-reducing condition except using non-reducing
buffer in place of reducing buffer (Figure 7).
Fig. 7: SDS PAGE analysis of Serratiopeptidase in microspheres
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Zeta potential
The microspheres were suspended in Phosphate buffer (pH 1.2) for 30 minutes. The
suspension (2% w/v) was employed for the determination of zeta potential. The results are
presented in Table 3.
Table 3: Zeta potential of selected microsphere formulations
Formulation Zeta potential (V)
A1
D2
Void Microspheres
+ 13.2
+ 8.5
+ 40.0
Mucoadhesivity
Mucoadhesivity was determined by an in situ method as described by [4]. A freshly cut
5-6 cm long piece of small intestine of rat was obtained and cleaned by washing with isotonic
saline. The piece was reversed with steel rod and the mucosal surface was exposed. Known
weights of microspheres were suspended in test tube containing phosphate buffer (pH 7.4) and
intestine section (exposed mucosal surface) was introduced into the suspension with a thread.
The intestinal piece was maintained at 80% relative humidity for 30 min in a desicator at 37C.
The piece was taken out and phosphate buffer pH 6.8 was allowed to flow over the intestinal
piece for about 2 minutes at a rate of 20 ml/min. The perfusate was collected and dried to
recover the unadhered particles. The % mucoadhesion was estimated by the ratio of amount
applied to adhered microspheres. The results are presented in Table 4.
Table 4: Percent mucoadhesion of various formulation of microspheres
Formulation % Mucoadhesion
A1
G2
D1
D2
36.95 3.66
40.13 0.10
19.16 5.85
31.66 1.66
Swelling properties
The water uptake by the non-crosslinked and crosslinked chitosan microspheres was
measured gravimetrically by swelling microspheres in deionized water (pH 6.0, 37°C) and
measuring changes in their weight during swelling. Microspheres were weighed and placed
inside a dialysis membrane, which was then introduced into the medium (50 ml) under
continuous stirring at 50 rpm and allowed to swell. The swollen samples were removed after
120 min and their surfaces were blotted with a filter paper to remove medium adsorbed on the
surface and then immediately weighed. Each swelling experiment was repeated twice and the
average value was taken as the degree of swelling, as calculated by:
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S.W. = Mt-Mo / t
where, Mt denotes the weight of the swollen sample at time t and Mo is the initial
weight of the sample before swelling. The results are presented in Table 5.
Table 5: Degree of swelling of coated formulation
Formulation Degree of swelling
A1
A2
A3
E1
E2
E3
G1
G2
G3
0.76 0.04
0.86 0.04
0.80 0.02
0.78 0.04
0.72 0.06
0.78 0.04
0.80 0.03
0.78 0.02
0.66 0.06
X-Ray Powder Diffractometry
X-ray powder diffractometry (XRD) was carried out to investigate the effect of
microencapsulation process on crystallinity of the drug. Powder XRD patterns were recorded on
a powder XRD using Ni-filtered, CuKα radiation, a voltage of 30 Kv, and a current of 15 mA. The
scanning rate employed was 1° min-1
over the 20° to 60° diffraction angle (2θ) range. The XRD
patterns of polymers, drug and drug-loaded microspheres were recorded (Figure 8).
Microspheres were triturated to get fine powder before taking the scan.
Fig. 8: X-ray powder diffractometry of serratiopeptidase, polymer, chitosan and microparticles.
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Statistical Analysis
Statistical significance of all the data generated was tested by analysis of variance
(ANOVA) followed by studentized range test. A confidence limit of P<0.05 was fixed for
interpretation of the results using the software PRISM (Graphpad, San Diego, CA).
RESULTS AND DISCUSSION
Some of the challenges of encapsulating proteins/enzymes for sustained delivery
namely high loading with high recovery, maintenance of enzyme integrity and sustained
delivery with a pH-responsive release have been successfully met with microspheres in this
study. It offers advantage for drug delivery of proteins/ enzymes, which can also be extended to
other therapeutic macromolecules.
Preparation of the microparticles
Chi-STP microspheres prepared by emulsification–solvent evaporation technique
showed good granulation. The particle shape was nearly spherical (Fig. 2). The mean particle
diameter was 8.3m. The mean STP content was between 13.7-31.2% (w/w), and the
encapsulation efficiency was more than 38%. ER-Chi-STP was prepared at different combination
ratios of Chi-STP to ER. The drug content was almost proportional to the ratio of Chi-STP.
Encapsulation efficiencies were 95.3–119.2%, indicating that the ideal drug content was well
achieved. Coated microspheres prepared by simple emulsification solvent evaporation method
with Chi-STP microspheres and Eudragit, showed a larger size, with the increase in amount of
Eudragit. The particle shape of A3, B3, etc. was irregular, but rugged ellipsoid.
Morphology of microspheres was examined by optical and s canning electron
microscopy. The view of the uncoated microspheres showed a hollow structure with a smooth
surface morphology whereas coated microspheres displayed a rough surface. The shell of the
uncoated microspheres showed some porous structures, which might have been caused by the
evaporation of solvent entrapped within the shell of microspheres after forming a smooth
dense skin layer. In contrast, the coated microspheres displayed continuous surface without
any porous structure.
The effect of the ratio of drug: polymer (Chitosan) in the aqueous phase on the
formation of the microspheres was evaluated keeping the volume of aqueous phase constant at
5 ml. The yield of the microspheres was found to be 85-98%. A variation in drug release was
observed, with highly loaded microspheres releasing more drugs. However, the average particle
size and the coating thickness increased as the amount of polymer were increased as reported
by Hori et al. [2]
The temperature of the dispersing medium was an important factor in the formation of
microspheres, because it controls the evaporation rate of the solvent. The microspheres
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October – December 2011 RJPBCS Volume 2 Issue 4 Page No. 673
prepared at low temperatures (35C), had irregular morphology. At higher temperatures
(45C), the microspheres prepared had a darker brownish color and uniform shape. The
optimum temperature to form good microspheres was found to be 35 and 40C.
Emulsification–solvent evaporation technique gave Chi-STP microspheres, which had
pores adequate for release of STP. The coating with ER prevented Chi-STP from the dissolution
in SGF (pH 1.2). ER-coated Chi-STP microspheres released STP very slowly in mixed phosphate
buffered saline (pH 6.8), than Chi-STP. It was observed that high entrapment efficiency was
achieved at optimum drug: polymer ratio and optimum temperature.
In vitro drug release
Release of drug from pH responsive microspheres was evaluated at pH 1.2, and pH 6.8.
The initial slow release at the entire tested medium may be due to coating with Eudragit L100.
The uncoated microspheres of chitosan alone released a significant amount of drug through the
pores of the microspheres, as the polymer was soluble in acidic pH whereas the coating
prevented the release of drug. A combination of polymer was used for current study to design
pH responsive delivery system, which released most of the drug at lower gastrointestinal tract.
As the amount of Eudragit L 100 used in the preparation was increased, the release of the drug
decreased. The release in the mixed phosphate buffer (pH 6.8) may be due to the porous
nature of chitosan and Eudragit L100 and increase in the pores of the microspheres due to
swelling of chitosan. As the drug was most soluble in pH 6.8 and 7.4, its release was highest in
these mediums caused by diffusion through pores. The release of drug in the mixed phosphate
buffer (pH 6.8) was many times elevated than at pH 1.2. At pH 6.8, nearly 50% of the drug was
released as observed within first 6 hours. It may be due to swelling of chitosan in mixed
phosphate buffer.
In case of formulation A1 coated with Eudragit L 100, the maximum drug release was
seen in pH 6.8 and minimum at pH 1.2. There was an initial fast release of drug in pH 6.8, which
may be due to the dissolution of Eudragit L 100, in alkaline pH.
Formulation A3 prepared by coating with polymer at highest ratio to microsphere, also
showed the maximum drug release at pH 6.8. The effect of coating ratio of polymer to
microsphere on dissolution time was in the sequence as1:1<1:2<1:5, dissolution time.
X-ray powder diffraction study
The x-ray powder diffraction patterns of microspheres along with those of physical
mixtures and raw crystals of drug and polymer were studied. The Serratiopeptidase-Chitosan-
Eudragit system, in the form of microsphere, indicated the presence of the crystalline STP but
with a dramatic decrease of the intensity of the signal because of both a dilution effect and a
decrease in crystallinity of the drug. These results were in compliance with the reports of [6].
Detection of drug by SDS-PAGE technique
Formulation A and A1 was identified and tested for purity by SDS-PAGE technique. The
presence of drug in the microsphere was confirmed by this technique.
Bioadhesive study
The bioadhesion of microspheres was measured by the method reported by Rao and
Buri [4]. The adsorption of microsphere to mucin is expected to be dominated by the
electrostatic attraction between the positively charged chitosan and negatively charged mucin.
Therefore, the surface charges of microspheres represented by zeta potential influenced the
amount bioadhesed. The amount of microsphere adsorbed increased with increasing chitosan
concentration. Also, in conformity with the electrostatic attraction theory, the amount of the
adsorption decreased with decreasing zeta potential. Microspheres with highest zeta potential
are highly adsorbed. The mucoadhession of the microspheres increased with increase in net
concentration of chitosan.
The water uptake of microspheres prepared at 40ºC and 45ºC was more than those
prepared at 35ºC. It also increased with increase in chitosan content.
CONCLUSION
The drug carriers developed for oral peptide delivery in the present study have been
tailored to protect the sensitive macromolecular drug from the harsh environment of the
stomach and deliver biologically active peptide for an extended period of time to more favorable
regions for absorption along the GI tract. The results indicated pH-responsive release of
serratiopeptidase and also confirmed integrity of drug within the formulation. The integrity of the
drug could be maintained during the formulation process and other formulation objectives i.e.,
high encapsulation yield and loading combined with pH responsive release of drugs were also
achieved. Further, the method of preparation of microspheres adopted i.e. the emulsification
process, is amenable to easy industrial scale-up. These findings corroborate the idea that the
combination of pH-responsive and mucoadhesive properties can be utilized effectively to devise
strategies for oral delivery of peptidal drugs.
Declaration of Interest
The authors report no conflict of interests. The authors alone are responsible for the
content and writing of the paper.
REFERENCES
[1] Bogataj M, Burjak M, Velnar M, Grabnar I, Mrhar A. Int J Pharm 2001; 224: 123-30.
[2] Hori M, Onishi H, Machida Y. Int J Pharm 2005; 297: 223-34.
[3] Moriya N, Nakata M, Nakamura M, Takaoka M, Iwasa S, Kato K, Kakinuma A. Biotechnol
Appl Biochem 1994; 20: 101-8.
[4] Rao KVR, Buri P. Int J Pharm 1989; 52: 265-70.
[5] Ryden AN, Hofsten BV. Acta Chemica Scandinavica 1968; 22: 2803-8.
[6] Tayade PT, Kale RD. AAPS PharmSci 2004; 6(1): E12.
October – December 2011 RJPBCS Volume 2 Issue 4 Page No. 662
Research Journal of Pharmaceutical, Biological and Chemical
Sciences
p
H
-Responsive and Mucoadherent Microparticles for Oral Delivery of
Serratiopeptidase: Development and In Vitro Characterization
Preeti K Suresh1
* and Anish Chandy2
1 University Institute of Pharmacy, Pt. Ravishankar Shukla University, Raipur (CG) INDIA
2 School of Pharmacy, Chouksey Engineering College, Bilaspur (CG) INDIA
ABSTRACT
The aim of the present study was to explore the potential of enteric coated chitosan microspheres as
carriers for oral site-specific delivery of peptides and proteins. In this study, microspheres of serratiopeptidase
were prepared by water in oil (w/o) emulsification solvent evaporation technique. Ratio of chitosan and
serratiopeptidase was varied in the range of 1:1, 1:2 and 2:1. The microspheres were then coated with Eudragit L
100. The product was obtained as non-aggregated free flowing microparticles. The drug content was found to be
between 15 to 40%. The presence of protein drug in the microparticles was confirmed by SDS-PAGE technique. In
vitro drug release studies indicated pH-responsive release profile. In vitro release of the drug first into phosphate
buffer pH 1.2 for 1 hr and then in phosphate buffer pH 6.8 at 37ºC was determined as a function of crosslinking
coating density of the microparticle. The pH-responsive behavior of the microparticle was confirmed, as there was
less than 10% release in phosphate buffer pH1.2. The extent of drug release had a remarkable dependence on the
coating density of the Eudragit L 100, the highly crosslinked spheres releasing only around 35% of the incorporated
drug in 6 hour compared to 40% from lightly coated spheres. The mucoadhesive nature of the microparticles was
established by an in situ method. These results indicate that Eudragit L 100 coated chitosan microparticles are
promising carriers for pH-responsive oral drug delivery of peptides and proteins.
Keywords: peptide, Eudragit L 100, Chitosan, pH-dependent, pH-sensitive, intestine-specific.
*Corresponding author
Email: preeti_venugopalan@yahoo.co.inINTRODUCTION
The intestinal drug targeting has been exploited for systemic delivery of active drugs. Most of the
peptide and protein drugs are unstable in the stomach and upper part of intestine. Apart from stability
problems, peptides are not well absorbed from the lumen of the GIT due to their large molecular size and
have high sensitivity to brush border peptidase activity. Comparatively, proteolytic activity of intestinal
mucosa is less than that observed in the stomach. Intestinal specific drug delivery systems protect peptide
drugs from hydrolysis and enzymatic degradation in the stomach, and eventually release drugs in the
jejunum, deuodenum, ileum or colon, which promises greater systemic bioavailability.
Serratiopeptidase (STP) is an anti-inflammatory, proteolytic enzyme isolated from the
microorganism, Serratia sp. HY-6 and is orally active [3]. Oral proteolytic enzymes have been
used successfully for inflammatory conditions. Recently, the intestinal absorption of orally
administered STP has also been demonstrated. To achieve an ideal therapeutic effect, however,
it is essential that any enzyme preparation be enterically coated so as to release the enzymes in
the intestines (where they can be absorbed) and not in the stomach (where they can be
digested). STP degrades in the stomach, but it is well absorbed from the small intestine.
Microsphere based drug delivery systems have received considerable attention in
recent years. The most important characteristic of microspheres is the microphase separation
morphology, which endows it with a controllable variability in degradation rate and drug
release.
pH-activated drug delivery systems permit the drug release at specific pH and the drug
molecules can thus be protected from degradation in the hostile gastric environment and in the
intestinal fluid the polymers dissolve and release the drug at intestinal pH>6.8. These types of
systems can be fabricated with polymers which are insoluble in acidic pH of stomach, but
soluble in basic pH prevailing in the intestine, viz., Eudragit L100, Eudragit S, Eudragit L, HMCP,
etc.
The aim of the present of work was to achieve site-specific controlled delivery of STP in
the small intestine. To achieve this objective Eudragit coated chitosan microparticles of STP were
prepared. The Eudragit coating will dissolve in the small intestine leaving chitosan microsphere,
which swells in the intestinal fluid leading to controlled release of STP from the microspheres.
The proposed mechanism is illustrated in Figure 1.
Fig. 1: Proposed mechanism of Eudragit®
coated chitosan microsphereshttp://www.rjpbcs.com/pdf/2011_2(4)/[70].pdf
ISSN: 0975-8585
October – December 2011 RJPBCS Volume 2 Issue 4 Page No. 664
MATERIAL AND METHODS
Materials
Chitosan (degree of deacetylation, 85.2%) particles with a viscosity 50 cps was obtained
as a gift sample from CFTI, Kochi, India. Serratiopeptidase was obtained from Centaur Labs Ltd,
Mumbai, India, as a gift sample. All other reagents used were of analytical grade from different
commercial manufacturers.
Development of Microparticles
Preparation of STP-containing chitosan microspheres (Chi-STP)
Chitosan (Chi) microspheres containing STP were prepared using w/o emulsification–
solvent evaporation method as reported by Hori et al [2]. Chitosan (100 mg) was dissolved in 5
ml of a 1% (v/v) acetic acid aqueous solution, and to this 2 ml of aqueous solution containing
STP (100 mg) was added. The chitosan (Chi) solution containing STP was dripped over 5 min into
200 ml of liquid paraffin containing SO-15
®
at 1% (w/v), which was continuously stirred at 500
rpm. The w/o emulsion obtained was stirred vigorously at 35C for 24 h, diethyl ether was
added, and the mixture was further, stirred overnight. The particles obtained were washed with
diethyl ether, 25% (w/w) ammonia aqueous solution, ethanol and diethyl ether in that order.
The particles were obtained by filtration and dried in air to yield microspheres.
ER-coated Chi-STP microspheres
Eudragit®
(ER)-coated Chi-STP microspheres (ER-Ch-STP) were prepared by coating Chi-
STP with Eudragit L 100 following the method reported by Bogataj et al [1]. Eudragit L 100 was
dissolved in 20 ml of acetone and the chitosan microspheres were dispersed in it. Then
equivalent amount of magnesium stearate was added and the dispersion was added dropwise
to 100 ml liquid paraffin (Heavy: Light = 1:1). To this dispersion 20 ml hexane was added with
stirring. The mixture was stirred for 90 min and the microspheres formed were collected by
filtration and washed with hexane twice and dried at room temperature.
Optimization of Process Variables
Various process variables, which could affect the preparation and properties of the
microspheres, were identified and studied. Preparation of pH-responsive microspheres involves
various process variables out of which drug: polymer ratio (D), temperature (T) and coating
polymer: microsphere ratio (P) was selected for the optimization of formulation.
The formulation code and respective variables used in the preparation of microspheres
are listed in Table 1. The effect of these variables was observed on particle size and size
distribution, % drug entrapment and % yield of microspheres.
CHARACTERIZATION
Size distribution and morphology
Scanning Electron Microscopy (SEM JEOL JSM-5800, Japan) was used to evaluate the
surface texture, shape and size of the microspheres. The samples for SEM were prepared by
lightly sprinkling the microspheres powder on a double adhesive tape, which stuck to an
aluminum stub. The stubs were then coated with gold to a thickness of about 300A using a
sputter container. The photomicrographs are shown in Figures 2 and 3.
Yield
To determine the % yield, weight of prepared microspheres was divided by the total
weight of all the non-volatile components used for the preparation of the microspheres. The
results are presented in Table 2.
Drug content
Chi-STP microspheres (10 mg) were dissolved in 10 ml of SIF (pH 7.4) by stirring with a
vortex mixer. An aliquot of 150 μl of the solution was treated with casein solution to detect itsproteolytic activity and to measure the amount of STP from the standard curve. The resultant
solution was centrifuged at 3000 rpm for 5 min and the supernatant was analyzed
spectrophotometrically at 280 nm.
Table 2: Encapsulation efficiency, yieldIn vitro release profile
Accurately weighed (60 mg) microspheres equivalent to 10 mg drug were suspended in
500 ml of mixed phosphate buffered saline (pH 6.8), and incubated at 37o
C and 90 rpm. After 1,
2, 3, 4, 5, 6 and 24 h incubation, 1ml of medium was collected, 4.5 ml Casein solution added
and allowed to stand for 37o
C for 20 min. [5] To the above solution, and trichloroacetic acid was
added to break the reaction. The supernatant collected and was used for the quantitative assay
of STP. For this assay, the final sample was obtained after centrifugation at 10,000 rpm for 5
min, and absorbance was measured spectrophotometrically at 280
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ER-Chi-STP accurately weighed microspheres equivalent to 10mg drug were suspended
in 500 ml of phosphate buffered saline (pH 1.2) and incubated at 37o
C at 90 rpm. The samples
were collected at regular intervals of 15 min. The pH was then adjusted to pH 6.8. At 1, 2, 3, 4,
5, 6 and 24 h after the start of the incubation in mixed phosphate buffered saline (pH 6.8), 1ml
of the medium was taken, 4.5 ml casein solution was added and allowed to stand at 37o
C for 20
min. Trichloroacetic acid was added to this solution to break the reaction and then centrifuged
at 10000 rpm for 10 min. The supernatant was used for the measurement of the amount of STP
released. Proteolytic activity was determined to measure the concentration of STP by
spectrophotometric estimation at 280 nm. The results are presented in Figures 4-6.
Fig. 4: Drug release profile of various microparticles (drug: polymer ratio of 1:1)
Fig. 5: Drug release profile of various microparticles (drug: polymer ratio of 1:2)
Detection of Drug by SDS PAGE
10 mg microspheres were dissolved in 1ml phosphate buffered saline (pH 7.4), then
vortexed for 10 min, allowed to stand for 5 min and again vortexed for 5 min to obtain
equilibrium concentration of drug in solution. The sample was centrifuged at 10,000 rpm for 10
min. Supernatant was collected and used for SDS- PAGE. 20µl of supernatant was taken and
applied on gel with reducing buffer and dye methylene blue. The stain was detected by marker
silver stain. This procedure was repeated for non-reducing condition except using non-reducing
buffer in place of reducing buffer (Figure 7).
Fig. 7: SDS PAGE analysis of Serratiopeptidase in microspheres
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Zeta potential
The microspheres were suspended in Phosphate buffer (pH 1.2) for 30 minutes. The
suspension (2% w/v) was employed for the determination of zeta potential. The results are
presented in Table 3.
Table 3: Zeta potential of selected microsphere formulations
Formulation Zeta potential (V)
A1
D2
Void Microspheres
+ 13.2
+ 8.5
+ 40.0
Mucoadhesivity
Mucoadhesivity was determined by an in situ method as described by [4]. A freshly cut
5-6 cm long piece of small intestine of rat was obtained and cleaned by washing with isotonic
saline. The piece was reversed with steel rod and the mucosal surface was exposed. Known
weights of microspheres were suspended in test tube containing phosphate buffer (pH 7.4) and
intestine section (exposed mucosal surface) was introduced into the suspension with a thread.
The intestinal piece was maintained at 80% relative humidity for 30 min in a desicator at 37C.
The piece was taken out and phosphate buffer pH 6.8 was allowed to flow over the intestinal
piece for about 2 minutes at a rate of 20 ml/min. The perfusate was collected and dried to
recover the unadhered particles. The % mucoadhesion was estimated by the ratio of amount
applied to adhered microspheres. The results are presented in Table 4.
Table 4: Percent mucoadhesion of various formulation of microspheres
Formulation % Mucoadhesion
A1
G2
D1
D2
36.95 3.66
40.13 0.10
19.16 5.85
31.66 1.66
Swelling properties
The water uptake by the non-crosslinked and crosslinked chitosan microspheres was
measured gravimetrically by swelling microspheres in deionized water (pH 6.0, 37°C) and
measuring changes in their weight during swelling. Microspheres were weighed and placed
inside a dialysis membrane, which was then introduced into the medium (50 ml) under
continuous stirring at 50 rpm and allowed to swell. The swollen samples were removed after
120 min and their surfaces were blotted with a filter paper to remove medium adsorbed on the
surface and then immediately weighed. Each swelling experiment was repeated twice and the
average value was taken as the degree of swelling, as calculated by:
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S.W. = Mt-Mo / t
where, Mt denotes the weight of the swollen sample at time t and Mo is the initial
weight of the sample before swelling. The results are presented in Table 5.
Table 5: Degree of swelling of coated formulation
Formulation Degree of swelling
A1
A2
A3
E1
E2
E3
G1
G2
G3
0.76 0.04
0.86 0.04
0.80 0.02
0.78 0.04
0.72 0.06
0.78 0.04
0.80 0.03
0.78 0.02
0.66 0.06
X-Ray Powder Diffractometry
X-ray powder diffractometry (XRD) was carried out to investigate the effect of
microencapsulation process on crystallinity of the drug. Powder XRD patterns were recorded on
a powder XRD using Ni-filtered, CuKα radiation, a voltage of 30 Kv, and a current of 15 mA. The
scanning rate employed was 1° min-1
over the 20° to 60° diffraction angle (2θ) range. The XRD
patterns of polymers, drug and drug-loaded microspheres were recorded (Figure 8).
Microspheres were triturated to get fine powder before taking the scan.
Fig. 8: X-ray powder diffractometry of serratiopeptidase, polymer, chitosan and microparticles.
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Statistical Analysis
Statistical significance of all the data generated was tested by analysis of variance
(ANOVA) followed by studentized range test. A confidence limit of P<0.05 was fixed for
interpretation of the results using the software PRISM (Graphpad, San Diego, CA).
RESULTS AND DISCUSSION
Some of the challenges of encapsulating proteins/enzymes for sustained delivery
namely high loading with high recovery, maintenance of enzyme integrity and sustained
delivery with a pH-responsive release have been successfully met with microspheres in this
study. It offers advantage for drug delivery of proteins/ enzymes, which can also be extended to
other therapeutic macromolecules.
Preparation of the microparticles
Chi-STP microspheres prepared by emulsification–solvent evaporation technique
showed good granulation. The particle shape was nearly spherical (Fig. 2). The mean particle
diameter was 8.3m. The mean STP content was between 13.7-31.2% (w/w), and the
encapsulation efficiency was more than 38%. ER-Chi-STP was prepared at different combination
ratios of Chi-STP to ER. The drug content was almost proportional to the ratio of Chi-STP.
Encapsulation efficiencies were 95.3–119.2%, indicating that the ideal drug content was well
achieved. Coated microspheres prepared by simple emulsification solvent evaporation method
with Chi-STP microspheres and Eudragit, showed a larger size, with the increase in amount of
Eudragit. The particle shape of A3, B3, etc. was irregular, but rugged ellipsoid.
Morphology of microspheres was examined by optical and s canning electron
microscopy. The view of the uncoated microspheres showed a hollow structure with a smooth
surface morphology whereas coated microspheres displayed a rough surface. The shell of the
uncoated microspheres showed some porous structures, which might have been caused by the
evaporation of solvent entrapped within the shell of microspheres after forming a smooth
dense skin layer. In contrast, the coated microspheres displayed continuous surface without
any porous structure.
The effect of the ratio of drug: polymer (Chitosan) in the aqueous phase on the
formation of the microspheres was evaluated keeping the volume of aqueous phase constant at
5 ml. The yield of the microspheres was found to be 85-98%. A variation in drug release was
observed, with highly loaded microspheres releasing more drugs. However, the average particle
size and the coating thickness increased as the amount of polymer were increased as reported
by Hori et al. [2]
The temperature of the dispersing medium was an important factor in the formation of
microspheres, because it controls the evaporation rate of the solvent. The microspheres
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prepared at low temperatures (35C), had irregular morphology. At higher temperatures
(45C), the microspheres prepared had a darker brownish color and uniform shape. The
optimum temperature to form good microspheres was found to be 35 and 40C.
Emulsification–solvent evaporation technique gave Chi-STP microspheres, which had
pores adequate for release of STP. The coating with ER prevented Chi-STP from the dissolution
in SGF (pH 1.2). ER-coated Chi-STP microspheres released STP very slowly in mixed phosphate
buffered saline (pH 6.8), than Chi-STP. It was observed that high entrapment efficiency was
achieved at optimum drug: polymer ratio and optimum temperature.
In vitro drug release
Release of drug from pH responsive microspheres was evaluated at pH 1.2, and pH 6.8.
The initial slow release at the entire tested medium may be due to coating with Eudragit L100.
The uncoated microspheres of chitosan alone released a significant amount of drug through the
pores of the microspheres, as the polymer was soluble in acidic pH whereas the coating
prevented the release of drug. A combination of polymer was used for current study to design
pH responsive delivery system, which released most of the drug at lower gastrointestinal tract.
As the amount of Eudragit L 100 used in the preparation was increased, the release of the drug
decreased. The release in the mixed phosphate buffer (pH 6.8) may be due to the porous
nature of chitosan and Eudragit L100 and increase in the pores of the microspheres due to
swelling of chitosan. As the drug was most soluble in pH 6.8 and 7.4, its release was highest in
these mediums caused by diffusion through pores. The release of drug in the mixed phosphate
buffer (pH 6.8) was many times elevated than at pH 1.2. At pH 6.8, nearly 50% of the drug was
released as observed within first 6 hours. It may be due to swelling of chitosan in mixed
phosphate buffer.
In case of formulation A1 coated with Eudragit L 100, the maximum drug release was
seen in pH 6.8 and minimum at pH 1.2. There was an initial fast release of drug in pH 6.8, which
may be due to the dissolution of Eudragit L 100, in alkaline pH.
Formulation A3 prepared by coating with polymer at highest ratio to microsphere, also
showed the maximum drug release at pH 6.8. The effect of coating ratio of polymer to
microsphere on dissolution time was in the sequence as1:1<1:2<1:5, dissolution time.
X-ray powder diffraction study
The x-ray powder diffraction patterns of microspheres along with those of physical
mixtures and raw crystals of drug and polymer were studied. The Serratiopeptidase-Chitosan-
Eudragit system, in the form of microsphere, indicated the presence of the crystalline STP but
with a dramatic decrease of the intensity of the signal because of both a dilution effect and a
decrease in crystallinity of the drug. These results were in compliance with the reports of [6].
Detection of drug by SDS-PAGE technique
Formulation A and A1 was identified and tested for purity by SDS-PAGE technique. The
presence of drug in the microsphere was confirmed by this technique.
Bioadhesive study
The bioadhesion of microspheres was measured by the method reported by Rao and
Buri [4]. The adsorption of microsphere to mucin is expected to be dominated by the
electrostatic attraction between the positively charged chitosan and negatively charged mucin.
Therefore, the surface charges of microspheres represented by zeta potential influenced the
amount bioadhesed. The amount of microsphere adsorbed increased with increasing chitosan
concentration. Also, in conformity with the electrostatic attraction theory, the amount of the
adsorption decreased with decreasing zeta potential. Microspheres with highest zeta potential
are highly adsorbed. The mucoadhession of the microspheres increased with increase in net
concentration of chitosan.
The water uptake of microspheres prepared at 40ºC and 45ºC was more than those
prepared at 35ºC. It also increased with increase in chitosan content.
CONCLUSION
The drug carriers developed for oral peptide delivery in the present study have been
tailored to protect the sensitive macromolecular drug from the harsh environment of the
stomach and deliver biologically active peptide for an extended period of time to more favorable
regions for absorption along the GI tract. The results indicated pH-responsive release of
serratiopeptidase and also confirmed integrity of drug within the formulation. The integrity of the
drug could be maintained during the formulation process and other formulation objectives i.e.,
high encapsulation yield and loading combined with pH responsive release of drugs were also
achieved. Further, the method of preparation of microspheres adopted i.e. the emulsification
process, is amenable to easy industrial scale-up. These findings corroborate the idea that the
combination of pH-responsive and mucoadhesive properties can be utilized effectively to devise
strategies for oral delivery of peptidal drugs.
Declaration of Interest
The authors report no conflict of interests. The authors alone are responsible for the
content and writing of the paper.
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[3] Moriya N, Nakata M, Nakamura M, Takaoka M, Iwasa S, Kato K, Kakinuma A. Biotechnol
Appl Biochem 1994; 20: 101-8.
[4] Rao KVR, Buri P. Int J Pharm 1989; 52: 265-70.
[5] Ryden AN, Hofsten BV. Acta Chemica Scandinavica 1968; 22: 2803-8.
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