Drug Development and Industrial Pharmacy, 34:181–188, 2008

Copyright © Informa Healthcare USA, Inc.

ISSN: 0363-9045 print / 1520-5762 online
DOI: 10.1080/03639040701539479

Development and In Vitro Evaluation of Alginate

LDDI

Gel–Encapsulated, Chitosan-Coated Ceramic

Nanocores for Oral Delivery of Enzyme
Manju Rawat, Deependra Singh, Shailendra Saraf, and Swarnlata Saraf
Alginate Gel–Encapsulated, Chitosan-Coated Ceramic Nanocores

Institute of Pharmacy, Pt Ravishankar Shukla University, Raipur (C.G.), India

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and protein drugs (Zhou & Li, 1991b). But many of them
The successful administration of protein and peptide drugs by require special formulation technologies to overcome drug-
oral route maintaining their active conformation remains a key associated problems such as chemical and physical instability,
challenge in the field of pharmaceutical technology. In the present poor bioavailability, and potentially strong side effects requir-
study, we propose the use of a nanosize ceramic core-based system
for effective oral delivery of acid-labile model enzyme, serratiopep-
ing drug enrichment at the site of action.
tidase (STP). Ceramic core was prepared by colloidal precipitation Proteolytic enzymes represent an important class of protein
and sonication of disodium hydrogen phosphate solution and cal- and peptides with primary pharmacological use as anti-inflam-
cium chloride solution at room temperature. The core was coated matory and digestive agents. One of the clearest indications of
with chitosan under constant stirring and Fourier-Transform Infra the general recognition of this premise is the vast annual
Red Spectroscopy (FTIR) confirmed phosphoric groups of calcium
expenditure of the pharmaceutical industry on exploring the
For personal use only.

phosphate linked with ammonium groups of chitosan in the nano-

particles; then the enzyme was adsorbed over the preformed nano- involvement of peptidases in human health and disease (Barret,
core. Protein-loaded nanocore was further encapsulated into 1991). Among this category, serratiopeptidase (STP) offers a
alginate gel for enzyme protection. Prepared system was character- powerful treatment for pain and inflammation with widespread
ized for size, shape, loading efficiency, and in vitro release profile use in arthritis, fibrocystic breast disease, chronic bronchitis,
(pH 1.2 and pH 7.4). The effect of processing variables on the size of
the core was evaluated to form small, uniform, and discrete nano-
and carpal tunnel syndrome (Kee, Tan, Lee, & Salmon, 1989;
cores. Stability and integrity of enzyme during processing steps was Majima, 1990).
assessed by in vitro proteolytic activity. The prepared system was These are normally delivered by the parenteral adminis-
examined to be spherical in shape with diameter 925 ± 6.81 nm tration. However complications such as thromboflebitis or
using TEM. The in vitro release data followed the Higuchi model, tissue necrosis and poor patient compliance have stimulated
showing a low amount (26% ± 2.4%) of diffusion-controlled drug
release (R2 = 0.9429) in acidic buffer up to a period of 2 to 6 hours,
the investigation of non-parenteral routes (Zhou, 1994).
signifying the integrity of alginate gel in acid. In the alkaline Among non-parenteral routes, oral administration is usually
medium sustained and nearly complete first order release of pro- preferred because it is most acceptable and convenient for
tein was observed up to a 6 hours. It is inferred that the protein- the patient. But oral bioavailability of these peptide drugs is
loaded ceramic core acts as a reservoir of the adsorbed enzyme and generally very low, owing to the acidic conditions of the
alginate gel provides protection to STP for controlled release in
intestinal pH when compared to the enzyme solution.
stomach, proteolytic activity of the gastrointestinal tract,
and poor permeability across intestinal mucosa (Zhou & Li,
Keywords ceramic nanocore; chitosan; serratiopeptidase; oral
1991a). Various approaches have been proposed to over-
delivery; proteolytic activity; alginate; peptide come the biopharmaceutical limitations associated with
these drugs, such as inhibition of the enzymatic degradation
(Morimoto et al., 1991), chemical modification of the pro-
tein (Conradi, Hilghers, Ho, & Burton, 1992), in situ gel
INTRODUCTION system (Shah & Paradkar, 2005), and the formulation of
Recent advancements in biotechnology and genetic research polymer-based carrier systems (Torchilin, Tischenko,
have led to an increased surge of interest in the use of peptide Smirnov, & Chazov, 1977).
Application of nanocarriers for drug delivery, especially
bioactive drugs, is an expanding area of research (Rawat,
Address correspondence to Dr. Swarnlata Saraf, Institute of Singh, Saraf, & Saraf, 2006) that provided the design of biom-
Pharmacy, Pt Ravishankar Shukla University, Raipur (C.G.), India aterials with controlled rates of drug release (Prokop, Kozlov,
492010. E-mail: [email protected] Newman, & Newman, 2002). It is tempting to expand the

181
 182 M. RAWAT ET AL.

utility of nanocarriers for delivery of therapeutic enzymes, times with 50 mL distilled water to remove even traces of
because particles larger than 20 μm are prone to be washed out, sodium chloride formed during the reaction. The precipitate
being inefficient for mucosal delivery (Lameiro, Lopes, was resuspended in distilled water and passed through a 0.2-
Martins, Alves, & Meloa, 2006). μm millipore filter to collect particles less than 0.2 μm.
But formulation processes must maintain the native three- Obtained calcium phosphate dihydrate core particles were dis-
dimensional structure and chemical integrity of the native pro- persed in chitosan solution (0.1% to 0.6% w/v) for effective
tein upon administration (Cleland, 1997). Thus the greatest coating under constant stirring and lyophilization. Three per-
limiting factor with these fragile modules as clinical tools is cent (3%) w/v sodium tripolyphosphate (STPP) solution was
due to the interaction between the drug carrier and the drug, added dropwise to the system as a cross-linking agent. Various
which affects physiochemical stability. Innovative techniques process parameters such as core to coat ratio (1:1 to 1:5), chito-
have led to the use of ceramics in high-tech applications. These san concentration (0.1% to 0.6% w/v), stirring speed (3,000 to
ceramic systems are capable of delivering chemicals and 12,000 g), and time (20 to 120 minutes) were optimized to get
biologicals effectively while maintaining their structural small, spherical, discrete coated nanoparticles. Obtained chito-
conformation and activity (Cherian, Rana, & Jain, 2000; san-coated ceramic nanocores were washed several times with
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Kossovsky, 1990). distilled water. Proteolytic enzyme STP was loaded into these
The aim of the present study is to design chitosan-coated CNC by soaking these particles in a fixed amount of STP solu-
nanocores (CNC) for effective loading and protection of an tion (0.1% w/v solution of STP in saline phosphate buffer, pH
active acid-labile large enzyme, STP. As an attempt towards 7.4) for 24 hours by constant stirring. These enzyme-loaded
increasing the bioavailability, conformational stability, and nanocarriers were mixed with sodium alginate solution (2% w/v).
activity of the enzyme, we have loaded STP into chitosan- To this, CaCl2 solution was added with Ca/alginate mass ratio
coated ceramic nanocores and encapsulated them into alginate 0.6% w/w for complete alginate gel formation (De & Robinson,
gel for effective protection, mucoadhesiveness, and sustained 2003) and stirred for 30 minutes to obtain particles encapsu-
release. In protein carrier selection, natural polymers such as lated in alginate gel. All operations were carried out at temper-
chitosan and alginate were selected because they are biosafe; ature below 4°C.
highly inert towards protein drugs; do not need organic sol-
For personal use only.

vents; and possess properties of mucoadhesiveness, biodegrad-

Morphology and Structural Characterization
ability, low toxicity, low immunogenicity, ready availability,
and inexpensiveness (Gombotz & Wee, 1998). In particular, The average size and size distribution of plain, protein-
morphology, loading, in vitro proteolytic activity, and release adsorbed chitosan-coated ceramic nanocores and alginate gel–
profile of prepared nanocarriers were evaluated as a function of encapsulated system were determined by transmission electron
the preparation procedure. microscopy (TEM, CM-12 Philips, Netherlands) after negative
staining with 1% phosphotungstic acid. Chitosan coating on
CNC was ascertained by FTIR. CNC were separated from the
MATERIALS AND METHODS suspension and dried by a freeze dryer (HETO Power
DryLL3000, Denmark), and their FTIR (Schimadzu FTIR-
Materials
8400S, Tokyo, Japan) was taken with KBr pellets.
STP (MW 52kDa) and chitosan (with 80% degree of
deacetylation and viscosity of 16 mPa) were received as gift
samples from Advanced Enzyme Technologies Ltd., Nasik, Enzyme Loading Efficiency
India, and Chemchito Natural Products, Chennai, India. Sodium The amount of enzyme loaded onto calcium phosphate dihy-
alginate and calcium chloride dihydrate were purchased from drate core was determined by a method reported by Loukas and
Loba Chemicals, Mumbai, India, and S. D. Fine Chemicals Gregoriadis (1997). Control formulations (without protein coat-
Ltd., Mumbai, India. Calcium phosphate was synthesized in the ing) were incubated under constant stirring with the known con-
laboratory. All other chemicals were of analytical grade. centration of drug for 24 hours at 4°C. The supernatant was
separated after centrifugation at 9,000 g for 1 hour below 4°C in
a refrigerated centrifuge (IV C1-6363, Remi Instruments,
Preparation of Alginate Gel–Encapsulated,
Mumbai, India). The protein remaining in the supernatant liquid
Chitosan-Coated Nanocores (ACNC)
after loading was estimated by measuring absorbance at 229.5 nm
Ceramic cores made up of calcium phosphate dihydrate by first derivative method spectrophotometerically (Shimadzu
were prepared in the laboratory by a procedure reported by UV-1700, Pharmaspec, Tokyo, Japan).
Kossovsky and colleagues (1990) with slight modifications. A
0.90 M solution of Na2HPO4 was slowly added to the 0.30 M
solution of calcium chloride and was sonicated (10 W) for In Vitro Release Studies
2 hours at 4°C. Precipitate of calcium phosphate was separated In order to visualize the release profile of the oral drug
by centrifugation at 7,200 g for 1 hour and then washed five delivery system, in vitro release profile of enzyme was carried
 ALGINATE GEL–ENCAPSULATED, CHITOSAN-COATED CERAMIC NANOCORES 183

out both in HCl (pH 1.2) and Phosphate Buffer Saline (PBS; (Kossovsky, 1990). During sonication, the nanosized NC self-
pH 7.4) buffer. One hundred (100) mg of enzyme-loaded carri- assemble due to increase in the surface free energy of calcium
ers were introduced into 10 mL of respective medium under phosphate particles (Kossovsky et al., 1996).
magnetic stirring (100 rpm) maintained at a temperature of 37° Ceramic cores were coated with the thin film of chitosan
± 0.5°C. At various time intervals, 0.1 mL of sample was with- solution (1% w/v acetic acid) by stirring at different speeds
drawn and replenished with fresh dissolution medium main- with 3% w/v STPP as cross-linking agent. STPP (3% w/v) was
tained at 37° ± 0.5°C. Samples withdrawn from acidic medium selected as it is a nontoxic polyanion capable of forming cross-
were centrifuged at 9,000 g for 10 minutes and the supernatant linked networks with chitosan. It specifically interacts with
was used for protein analysis. Samples obtained from phos- positively charged amine residues of chitosan with the degree
phate buffer were mixed with 0.2 M NaOH to raise pH above of cross-linking dependent on the concentration of STPP
7.0; then ethanol was added (50/50, v/v) to precipitate alginate. (Taqieddin, Lee, & Amiji, 2002).
Samples were then centrifuged and analyzed similarly to the Various process variables, for example, chitosan concentra-
sample obtained at pH 1.2. tion, core-to coat ratio, stirring speed, and stirring time, were
studied to obtain uniformly sized spherical discrete CNC
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(Table 1). Chitosan was used in the concentration ranging from

In Vitro Proteolytic Activity
0.1% to 0.6% w/v with fixed core concentration (1% w/v). The
Prepared ACNC and plain STP solution were placed sepa- size of CNC increased with the increase in the chitosan con-
rately in HCl buffer (pH 1.2) or phosphate buffer (pH 7.4) centration up to 0.5% w/v. Kawashima, Yamamoto, Takeuchi,
maintained at 37° ± 0.5°C and stirred constantly at 100 rpm.
After 2 hours, protein was recovered as reported previously for
release studies. Samples were then assayed for proteolytic
activity (n = 3). TABLE 1
The proteolytic activity was determined as per the method Effect of Process Parameters on the Particle Size of the CNC
reported in Food Chemical Codex (2003). The assay was based
on a 30-minute proteolytic hydrolysis of casein at 37°C and pH S. No. Observation Size (nm)
For personal use only.

7.0. Unhydrolyzed casein was removed by filtration and the

a) Core-to-Coat Ratio
solubilized casein was determined spectrophotometrically at a
1. 1:1 Irregular 162 ± 1.65
wavelength of 275 nm. In this method, the protease activity is
2. 1:2 Irregular 274 ± 1.43
expressed as protease unit (PC) units of preparation derived
3. 1:3 Non-uniform 396 ± 6.54
from Bacillus subtilis var. and Bacillus licheniformis var. One
spherical
bacterial protease unit (PC) is defined as quantity of enzyme
4. 1:4* Spherical 410 ± 4.32
that produces 1.5 μg/mL equivalent of L-tyrosine per minute
5. 1:5 Spherical 412 ± 4.34
under the condition of the assay.
Activity of enzyme was calculated by equation: b) Stirring Time (minutes)
1. 20 Large irregular 784 ± 4.42
PC ⎛ Au ⎞ ⎛ 0.08 ⎞ 2. 40 Large discrete 718 ± 4.23
=⎜ ⎟⎜ ⎟ 3. 60 Moderately 645 ± 2.31
g ⎝ As ⎠ ⎝ 30 w ⎠
discrete
4. 80 Small spherical 542 ± 3.12
5. 100* Discrete small 419 ± 3.26
Au is the value obtained by subtracting blank reading from test
spherical
reading; As the absorption of standard solution; 0.08 the final
6. 120 Aggregates 684 ± 3.24
volume in mL of reaction mixture; 30 the time of the reaction
in minutes; and w the weight of the original sample in g. c) Stirring Speed (g)
1. 3,000 Large discrete 982 ± 6.78
Statistical Analysis 2. 6,000 Comparatively 762 ± 8.84
The results were expressed as mean ± standard deviation. small
Statistical analysis was carried out by student t-test, and statis- 3. 9,000* Small spherical 412 ± 3.58
tical significance was designated as p < .05 (SPSS). 4. 12,000 Distorted spherical 678 ± 6.89
aggregates
RESULTS AND DISCUSSION Chitosan concentration used was 0.5% w/v. Values are shown
The calcium phosphate dihydrate ceramic nanocores (NC) as representative of M ± SD for three independent determinations
were prepared by slight modification of reported method (p < .05). *variables selected for study.
 184 M. RAWAT ET AL.

and Kuno (2000) also supported the same result. Chitosan con- 1200
centrations above 0.5% w/v, for example, 0.6% w/v, resulted

Particle size (nm)

1000
in aggregated mass, which may be due to increased viscosity of
800
chitosan solution. So the concentration of chitosan selected
was 0.5% w/v. The effect of core-to-coat ratio from 1:1 to 1:5 600
on the size of CNC showed that the size of the nanocores 400
increased from 162 ± 1.65 nm to 412 ± 4.34 nm (Figure 1 and 200
Table 1). The size of the coated particles increased up to 1:4 0
core-to-coat ratio, and then no significant increase in size was 0 3000 6000 9000 12000
observed, which is in agreement with results of Cherian and Stirring speed (g)
colleagues (2000). Moreover, highly aggregated forms were
FIGURE 3. Effect of stirring speed on the size of CNC. Results are given as
observed at this concentration, which could be due to the satu-
M ± SD (p < .05).
ration of the free surfaces of the core with the coating material.
Within the range of stirring time (20 to 120 minutes) investi-
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gated, the size and shape of the CNC changed from irregular
large particles (784 ± 4.42 nm) to comparatively small uniform stirring speed were selected to get spherical, chitosan-coated,
particles (419 ± 3.26 nm); at 120 minutes, larger particles were discrete nanoparticles, as can be seen through TEM photo-
observed, which might be due to aggregation of small particles graphs (Figure 4).
leading to increase in size (Figure 2). Similarly, the size Chitosan coating on the ceramic cores could not be clearly
decreased significantly from 982 ± 6.78 nm to 412 ± 3.58 nm identified in the TEM images. This could be due to close and
on increasing the speed of stirrer from 3,000 to 9,000 g (p < .05). longitudinal attachment of the CS chains during process
But on further increasing the speed to 12,000 g up to 100 minutes, required for TEM visualization (Garcia-Fuentes, Torres, &
increase in size with aggregated forms was observed. This may Alonso, 2005). CS coating results in 100% increase in the size
be due to increase in free surface energy, which increased the of ceramic cores due to the deposition of CS layers on the sur-
tendency of small particles to aggregate (Figure 3). Therefore, a face of the nanocores.
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1:4 core-to-coat ratio, 100 minutes stirring time, and 9,000 g as FTIR spectra of chitosan-coated calcium phosphate
nanocores (CNC) and chitosan are shown in Figure 5. FTIR
spectra of chitosan represents characteristic band at 3,434/cm
due to –NH2 and –OH group stretching vibration in chitosan
500 matrix. In CNC a shift in peak from 3,434/cm to 3,399/cm was
Particle size (nm)

400
observed, previously attributed to enhanced hydrogen bonding
(Yu, Du & Zheng, 1999). In CNC the peak at 1,644/cm disap-
300
Series1 peared and a new sharp peak at 1,632/cm appeared, and the
200 1,604/cm peak of –NH2 bending vibration shifted to 1,536/cm.
100 This shift in peak of –NH2 group can be due to the linkage
0
between phosphoric group of calcium phosphate and ammo-
0 2 4 6 nium ion of chitosan. A similar result was obtained in the study
Core:Coat ratio of chitosan film treated with phosphate NaH2PO4 (Knaul,
Hudson, & Creber, 1999). Chitosan exhibits good film-forming
FIGURE 1. Effect of core-to-coat ratio on the size of prepared CNC. Results abilities and stabilized the core through ionic and noncovalent
are given as M ± SD (p < .05).
forces. TEM results and FTIR studies provided a clear evi-
dence of the chitosan coating due to increase in particle size
and shift in the peak heights, respectively.
1000 After the optimization of processing variables (0.5% w/v
Particle size (nm)

800 chitosan concentration, 1:4 core-to-coat ratio, 100-minute stir-

600 ring time, 9,000 g stirring speed) on the basis of size and shape
of CNC, the enzyme was allowed to adsorb on the CNC by a
400
partial adsorption technique. The increase in the size of nano-
200
cores was evident from the TEM study of STP-loaded CNC
0 (524 ± 4.46 nm) (Figure 4c; Table 2). Chitosan is soluble in a
0 20 40 60 80 100 120 140
weak acidic solution, and thus the use of organic solvent can be
Stirring time (min)
avoided, which is favorable for maintaining bioactivity of pro-
FIGURE 2. Effect of stirring time on the size of prepared CNC. Results are tein and peptide drugs. Moreover, the amino groups of chitosan
given as M ± SD (p < .05) are protonated in an acidic solution and the resultant soluble
 ALGINATE GEL–ENCAPSULATED, CHITOSAN-COATED CERAMIC NANOCORES 185

(A) (B)

0.5 μm
0.5 μm
(C)
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(D)
For personal use only.

0.5 μm 1 μm

FIGURE 4. TEM of various stages of nanocarriers; (A) Calcium phosphate nanocores (NC); (B) Chitosan-coated nanocores (CNC); (C) STP-adsorbed CNC;
(D) Alginate-encapsulated CNC (ACNC).

polysaccharide is positively charged, which can bind strongly

to negatively charged surfaces of proteins above its isoelectric
point (Janes, Calvo, & Alonso, 2001). Iso-electric point of the
STP obtained from Serratio marcescens is 6.1 and above this
pH it attains negative charge, which is suitable for its binding
with the positively charged chitosan surface (Salamone &
Wodzinski, 1997). These surface-adsorbed nanocores provide
conformational stabilization as well as a high degree of surface
exposure to proteins (Kossovsky et al., 1996; Paul & Sharma,
2001).
Enzyme-loaded CNC were encapsulated in alginate gel for
protection of STP from getting exposed to acidic pH in the
stomach (Coppi, Iannuccelli, Leo, Bernabei, & Cameroni,
2002). Alginate gel encapsulation resulted in an increase in the
size of ACNC from 524 ± 4.46 nm to 925 ± 6.81 nm.
The results of size analysis data for the various stages of
nanocarriers measured by TEM are given in Table 2. With
each additional coating of ceramic cores with the chitosan,
enzyme, and alginate consecutively resulted in the increase in
size of nanocores, which is also evident from the TEM photo-
graphs (Figure 4).
FIGURE 5. FTIR spectra of (A) chitosan-coated calcium phosphate
nanocores and (B) pure chitosan. FTIR spectra indicate the clear shift in the
Enzyme loading efficiency of CNC (size 415 ± 4.26 nm)
peak height around 1,604/cm to 1,536/cm, indicating the linkage of phosphoric was determined and found to be 46.64% ± 1.48%. The size of
group of calcium phosphate with ammonium group of chitosan. STP-adsorbed CNC increased about 580 ± 4.46 nm due to
 186 M. RAWAT ET AL.

TABLE 2
Particle Size of Different Stages of Nanocores with Net Protein Loading
Core Enzyme-Adsorbed Alginate-Encapsulated Enzyme
S. No. Size (nm) CNC Ceramic Core Size (nm) System (nm) Loading (%)
1. 142 ± 4.26 415 ± 4.26 524 ± 4.46 925 ± 6.81 46.64 ± 3.48
All the values are representative of M ± SD for three independent determinations (p < .05).

adsorption of drug molecules on the surface of cores coated The transit time of a drug through the absorptive area of the
with 0.5% w/v chitosan solution. The increased protein pay- gastrointestinal tract (GIT) is between 9 and 12 hours (Gibaldi &
load with CS coated nanocores was observed due to the Perrier, 1982), whereas γ scintigraphy studies confirm a short
increase in size. GIT transit time from mouth to cecum of 4 to 6 hours (Shargel
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Proteolytic activity of alginate-encapsulated nanocarriers & Yu, 1999). Thus assuming a maximum GIT transit time of
was evaluated separately before and after treating them for 2 12 hours, a formulation in the cecum is expected to release its
hours in acidic (0.1N HCl, pH 1.2) and basic media (phosphate drug load within 6 hours. Considering the same, in vitro drug
buffer, pH 7.4); results are presented in Figure 6. The nanocar- release from the ACNC was studied for duration of 6 hours.
riers showed about 4.35% ± 0.08% loss of proteolytic activity Release of encapsulated protein in HCl buffer pH 1.2 was
in acidic medium whereas retention of activity in basic found to be significantly less than in PBS (pH 7.4). 99.6% ±
medium was found to be 98.26% ± 0.32%. Meager loss of 0.2% of the drug was released in PBS during a period of 6
activity in alkaline media may be due to processing steps hours (Figure 7). However, during the same period only
involved in preparation of formulation. At the same time plain 26.58% ± 2.4% of the drug was released in HCl buffer. Release
STP solution exhibited almost complete loss of activity in seems to be spontaneous in the alkaline pH, unlike alginate-
acidic medium and 86.84% activity was retained in alkaline encapsulated nanoparticles.
For personal use only.

medium. ACNC exhibited much better retention of proteolytic In order to investigate the release mechanism of present
activity compared with plain STP solution. Possible explanation drug delivery system, the release data of prepared ACNC in
for the improved physical and chemical stability of proteolytic acidic (pH 1.2) and alkaline (pH 7.4) buffer were fitted to clas-
enzyme in nanocarriers may be due to reduced mobilization of sic drug release kinetics models. The release rates were ana-
protein due to ionic association with the positively charged lyzed by least square linear regression method. Release models
polymer, that is, chitosan and alginate gel coating. such as first order model, Higuchi model, and Ritger-Peppas
empirical model were applied to the release data (Dredan,
Antal, & Racz, 1996; Peppas, 1985) (Table 3). The coefficient
of determination (R2) of equation for release of STP from
Proteolytic activity of plain and nanocarriers ACNC in alkaline buffer was 0.9664, whereas in acidic buffer
containning STP in acidic (pH 1.2) and alkaline buffer
(pH 7.4) it was 0.6502, signifying first order release pattern followed by
ACNC in the alkaline medium. There was an initial burst
120
release of 15.6% ± 0.24% and 25% ± 0.46% in the first hour in
% Proteolytic activity

100 both acidic and alkaline buffer, respectively. So the release

80
60
120
% cumulative release

40
100
20
80 acidic pH(1.2)
0
60
1 2
alkaline pH(7.4)
pH (1.2) pH (7.4) 40
20
plain STP solution Nanocarriers entrapped STP
0
0 2 4 6 8
FIGURE 6. Effect of acidic and alkaline pH on the in vitro proteolytic Time (hrs)
activity of plain STP solution and STP entrapped in ACNC. Proteolytic
activity was determined on the basis of 30-minute proteolytic hydrolysis of FIGURE 7. In vitro release profile of STP from optimized ACNC in
casein at 37°C and pH 7.0 as reported in food chemical codex (2003). Values acidic pH (1.2) and alkaline pH (7.4) buffer. Results are given as M ± SD
are shown as mean of three independent determinations. (p < .05).
 ALGINATE GEL–ENCAPSULATED, CHITOSAN-COATED CERAMIC NANOCORES 187

TABLE 3 disintegrated completely, releasing the entrapped core. But

Release Behavior of STP in Acidic and Alkaline Buffer because of the protein-adsorbed core structure, whole protein is
not released as a burst maintaining sustained effect. This result
ANC was attributable to the slight sustained release of drug signify-
S. No. Kinetics pH 1.2 pH 7.4 ing mixed type of release pattern.

1. First order
CONCLUSION
K 0.02303 0.9481
R2 0.6502 0.9664 ACNC appeared to be a promising system for the associa-
2. Higuchi tion and delivery of sensitive macromolecules such as proteins
(1–6 hr) K 6.7338 44.87 and peptides due to mild condition requirements for their for-
R2 0.7394 0.6925 mation. The prolonged activity was obtained due to slow
(2–6 hr) K 2.4378 12.796 release of the enzyme from the ACNC and the intact structure
R2 0.9429 0.8609 without denaturation or dehydration during delivery and stor-
age. The encouraging results obtained in this study could pro-
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3. Ritger-Peppas
(1–6 hr) n 0.2789 0.7123 pose ACNC for future in vivo studies, especially in the
R2 0.8088 0.7438 delivery of protein and peptide drugs. These novel nanocarriers
(2–6 hr) n 0.0916 0.1346 were found to be promising for protection of the spatial quali-
R2 0.9653 0.9026 ties for exhibiting better therapeutic effect. But further studies
in terms of pharmacokinetics, toxicology, and animal studies
K, release rate constant; R2, coefficient of determination; n, release are required for clinical utility of the formulation.
exponent.

ACKNOWLEDGMENT
data after 1 hour were fitted to Higuchi and Ritger-Peppas The authors are thankful to M/s Advanced Enzyme Tech-
equation. The value of coefficient of determination (R2) in nologies Ltd., Nasik, India, for the gift sample of STP; Chem-
For personal use only.

Higuchi equation was found to be 0.9429 and 0.8609 in acidic chito Natural Products, Chennai, India, for kind gift of
and alkaline buffer medium, respectively, which indicates the chitosan; SIF, AIIMS, New Delhi, India, for transmission elec-
integrity of alginate gel and diffusion-controlled release tron micrography; Director, Institute of Pharmacy, Pt. Ravis-
between 2 and 6 hours in acidic buffer, whereas in alkaline hankar Shukla University, Raipur (C.G.) India for providing all
buffer, R2 value doesn’t favor diffusion pattern. This difference necessary facilities for carrying out this work; and Chhattis-
in release pattern is due to pH sensitivity of alginate gel. At pH garh Council of Science and Technology (CCOST) for finan-
1.2, calcium alginate gel is converted to a unionized form of cial assistance.
alginic acid. Even if Ca+2 ions don’t contribute any more to sta-
bilization of the system leading to initial burst, alginate system
maintains its structure without any visible changes in morphol- REFERENCES
ogy (Zhou, Deng, & Li, 2001). This insight leads to dissocia- Barret, A. (1991). Peptidases: A view of classification and nomenclature. In:
V. Turk (Ed.), Proteases: A new perspective (pp. 1–56). Basel, Switzerland:
tion of ionic linkages and reduction in gel strength that may Birkhauser Verlag.
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