International Journal of Biological Macromolecules 50 (2012) 138–147

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International Journal of Biological Macromolecules

journal homepage: www.elsevier.com/locate/ijbiomac

Development of cloxacillin loaded multiple-unit alginate-based floating system

by emulsion–gelation method
Jadupati Malakar a , Amit Kumar Nayak b , Dilipkumar Pal c,∗
a
Bengal College of Pharmaceutical Science and Research, Durgapur-713212, West Bengal, India
b
Seemanta Institute of Pharmaceutical Sciences, Mayurbhanj-757086, Orissa, India
c
School of Pharmaceutical Sciences, IFTM University, Lodhipur Rajput, Delhi Road, Moradabad-244 102, U.P., India

a r t i c l e i n f o a b s t r a c t

Article history: This work investigates the development, optimization and in vitro evaluation of liquid paraffin-entrapped
Received 27 July 2011 multiple-unit alginate-based floating system containing cloxacillin by emulsion–gelation method for gas-
Received in revised form 7 October 2011 tro retentive delivery. The effect of process variables like drug to polymer ratio by weight, and liquid
Accepted 9 October 2011
paraffin to water ratio by volume on various physicochemical properties in case of liquid paraffin-
Available online 15 October 2011
entrapped calcium alginate beads containing cloxacillin applicable to drug entrapment efficiency, density
and drug release was optimized using 32 factorial design and analyzed using response surface method-
Keywords:
ology. The observed (actual values) responses were coincided well with the predicted values, given by
Calcium alginate
Liquid paraffin the optimization technique. The optimized beads showed drug entrapment efficiency of 64.63 ± 0.78%,
Beads density of 0.90 ± 0.05 g/cm3 , and drug release of 56.72 ± 0.85% in simulated gastric fluid (pH 1.2) after
Floating system 8 h with floating lag time of 8.45 min and floated well over 12 h in simulated gastric fluid (pH 1.2). The
Formulation parameters average size of all dried beads ranged from 1.73 ± 0.04 to 1.97 ± 0.08 mm. The beads were characterized
Optimization by SEM and FTIR for surface morphology and excipients–drug interaction analysis, respectively. All these
Response surface methodology beads showed prolonged sustained release of cloxacillin over 8 h in simulated gastric fluid (pH 1.2). The
cloxacillin release profile from liquid paraffin beads followed Korsmeyer–Peppas model over a period of
8 h with anomalous (non-Fickian) diffusion mechanism for drug release.
© 2011 Elsevier B.V. All rights reserved.

1. Introduction systems not only prolong the dosing intervals, but also increase
the patient compliance beyond the level of existing controlled
The oral route is considered as the most preferred route for release dosage forms. Several approaches have been reported in
administration of drugs due to low cost of therapy, ease of the literature for improved gastroretention of oral dosage forms
administration, patient compliance, etc. During past few decades, viz. floatation [4,5], mucoadhesion [6,7], sedimentation [8], unfold-
numerous oral controlled drug delivery systems have been inves- able, expandable, or swellable systems [9], superporous hydrogel
tigated and developed to act as drug reservoirs for release of drugs systems [10], magnetic systems [11], etc. Among them, floating
over a defined period of time at a controlled rate. The success of drug delivery is of particular interest for drugs which: (a) act locally
controlled oral drug deliveries is limited due to variable gastric in the stomach; (b) are primarily absorbed from the stomach; (c)
emptying time. The variable and too rapid gastrointestinal tran- are poorly soluble at an alkaline pH; (d) have a narrow window
sit can result in incomplete drug release from the dosage form at of absorption; (e) are unstable in the intestinal or colonic envi-
the absorption site in gastrointestinal tract lading to weaken effi- ronment [12]. Floating dosage forms are designed to be remained
cacy of the administered dose [1]. To overcome these limitations, buoyant on the gastric fluid because of its lower bulk density com-
various gastroretentive systems have been designed to be retained pared to that of the aqueous medium, thus retained in the stomach
in the gastric region for prolonged time and released incorporated for several hours and the drug is slowly at a desired rate [4]. Both
drug candidates, which enable sustained and prolonged input of the single-unit systems [13] and multiple-unit systems [1] have been
drug to the upper part of the gastrointestinal tract to obtain opti- reported in the literature. Multiple-unit floating drug delivery sys-
mal bioavailability of drug candidates [2,3]. These gastroretentive tems show several advantages over single-unit ones, among which:
avoiding all-or-nothing emptying, absence of impairing of perfor-
mance due to failure of a few units, more predictable drug release
∗ Corresponding author. Tel.: +91 3244243265; fax: +91 591 2360818; mobile: kinetics, less chances of localized mucosal damage and adminis-
+91 8126764064. tration of units with different release profiles or those containing
E-mail addresses: [email protected], [email protected] (D. Pal). incompatible substances [14].

0141-8130/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.ijbiomac.2011.10.001
 J. Malakar et al. / International Journal of Biological Macromolecules 50 (2012) 138–147 139

Among the various floatable multiple-unit dosage forms, cal- 2. Materials and methods
cium alginate gel beads have been developed in recent years as a
unique vehicle for multiple-unit drug delivery systems due to its 2.1. Materials
biocompatibility, biodegradability, reproducibility, simple method
of preparation, abundant sources, low cost and minimal process- Cloxacillin was purchased from B.S. Trader Pvt. Ltd., India.
ing requirements [15,16]. Gel beads of calcium alginate can be Sodium alginate (Central Drug House, India), calcium chloride
produced by extruding sodium alginate solution as droplets into (Merck Ltd., India), liquid paraffin (relative density = 0.84, Nice
calcium chloride solution. The hydrogel properties of calcium algi- Chemicals Pvt. Ltd., India) were used. All chemicals and reagents
nate beads have been proposed for controlling the release of used were of analytical grade.
various drug molecules [15]. Calcium alginate beads have also
been developed as various floating multiple-unit systems to pro- 2.2. Experimental design
long the gastro retention time [16,17]. However, various floating
calcium alginate beads suffered from rapid drug release, a prob- A two-factor, three-level factorial design (32 ) was employed for
lem, which was overcame by the addition of further additives [17]. optimization with drug to polymer ratio by weight (X1 ), and liq-
Buoyancy in the calcium alginate beads may be imparted by incor- uid paraffin to water ratio by volume (X2 ) as the prime selected
porating various low density oils such as mineral oils, olive oil, independent variables, which were varied at three levels, low level
sunflower oil, groundnut oil, castor oil, mentha oil, linseed oil, (−1), medium level (0), and high level (+1). The drug entrapment
etc. [16,18–20]. efficiency (%), density (g/cm3 ), and drug release at 8 h (%) used as
Cloxacillin is a ␤-lactam ring with an isoxazolyl side chain dependent variable (response). Design-Expert 8.0.3 software (Stat-
containing antibiotic and highly pencillinase as well as acid resis- Ease Inc., USA) was used for the generation and evaluation of the
tant. It is generally absorbed from oral route especially if taken statistical experimental design. The matrix of the design including
in empty stomach. But, plasma t1/2 of cloxacillin is about 1 h. It investigated responses i.e., drug entrapment efficiency (%), density
has maximum absorption in stomach due to its low pka value (g/cm3 ), and drug release after 8 h (%) in simulated gastric fluid (pH
(approx. 2.7). It has short biological half life of ∼2–3 h [21]. So, 1.2) are shown in Table 1.
the floating drug delivery of cloxacillin was planned to enhance For optimization, the effects of independent variables upon the
the gastric retention. However, reports are not available regard- responses were modelled using following second order polyno-
ing the gastroretentive delivery of cloxacillin in the previous mial equation involving independent factors and interaction factors
literature. was selected based on model analysis, lack of fit and R2 analy-
Designing controlled-release formulations with the minimum sis for various measured responses, studied in this investigation.
number of trials is very crucial for pharmaceutical scientists. The The effects of independent variables upon the drug entrapment
response surface methodology has been commonly used for opti- efficiency (%), density (g/cm3 ), and drug release after 8 h (%) in
mization of different pharmaceutical formulations [22,23]. Based simulated gastric fluid (pH 1.2) were modelled using following
on the design of experiments, response surface methodology quadratic mathematical model generated by 32 factorial design is
encompasses the generation of polynomial equations and responds following:
over the experimental domain to determine the optimum for-
mulation(s). The technique requires minimum experimentation
Y = b0 + b1 X1 + b2 X2 + b3 X1 X2 + b4 X12 + b5 X22
and time, thus providing to be far more effective and economi-
cal than the conventional methods of formulating dosage forms
[23]. The present investigation aims at developing liquid paraffin- where, Y is the response; b0 is the intercept, and b1 , b2 , b3 , b4 ,
entrapped calcium alginate beads containing cloxacillin using b5 are regression coefficients. X1 and X2 are individual effects; X12
ionotropically emulsion–gelation method as multiple-unit float- and X22 are quadratic effects; X1 X2 is the interaction effect. One-
ing delivery system. Computer aided optimization technique using way ANOVA was applied to estimate the significance of the model
a 32 factorial design was employed to investigate the effect of (p < 0.05). Individual response parameters were evaluated using the
two independent process variables (factors), i.e., drug to polymer F-test. The surface response plots, and contour plots were analyzed
ratio and liquid paraffin to water ratio on the properties of liquid to reveal the effect of independent factors (here drug to polymer
paraffin-entrapped calcium alginate beads containing cloxacillin ratio by weight and liquid paraffin to water ratio by volume) on
applicable to drug entrapment efficiency, density, and drug the measured responses (here drug entrapment efficiency, density,
release. and drug release after 8 h) were analyzed.

Table 1
Experimental plan of 32 full factorial design (coded values in bracket) with observed response values for different liquid paraffin-entrapped calcium alginate beads containing
cloxacillin.

Batch code Normalized levels of factors employed Responses

Drug to polymer Oil to water ratio Drug entrapment Density (g/cm3 )a Drug release after
ratio (by weight) (by volume) efficiency (%)a 8 h (%)a

F-1 0.25 (−1) 0.30 (−1) 66.11 ± 0.42 1.01 ± 0.04 40.26 ± 0.64
F-2 0.25 (−1) 0.50 (+1) 62.89 ± 0.74 1.01 ± 0.05 34.34 ± 0.70
F-3 0.25 (−1) 0.40 (0) 63.72 ± 0.83 1.01 ± 0.04 36.06 ± 0.38
F-4 0.38 (0) 0.50 (+1) 60.77 ± 0.57 0.82 ± 0.03 36.03 ± 0.42
F-5 0.50 (+1) 0.40 (0) 59.84 ± 0.43 0.79 ± 0.02 39.35 ± 0.75
F-6 0.38 (0) 0.40 (0) 62.84 ± 0.84 0.83 ± 0.03 38.23 ± 0.28
F-7 0.50 (+1) 0.50 (+1) 57.07 ± 0.76 0.74 ± 0.01 36.47 ± 0.64
F-8 0.50 (+1) 0.30 (−1) 64.17 ± 0.66 0.81 ± 0.02 46.31 ± 0.60
F-9 0.38 (0) 0.30 (−1) 65.45 ± 0.58 0.86 ± 0.03 44.98 ± 0.57
a
Mean ± S.D.; n = 3.
140 J. Malakar et al. / International Journal of Biological Macromolecules 50 (2012) 138–147

2.3. Preparation of liquid paraffin-entrapped calcium alginate of simulated gastric fluid (pH 1.2) maintained at 37 ± 0.5 ◦ C for 12 h
beads containing cloxacillin and the paddles were rotated at 50 rpm. The floating ability of the
beads was measured by visual observation. The time taken for the
Liquid paraffin-entrapped calcium alginate beads containing beads to float at the surface of the dissolution medium (known as
cloxacillin were prepared by emulsion–gelation method [18]. floating lag time) and duration of floating (buoyancy) for each batch
Briefly, required amount of sodium alginate was dissolved in of beads were noted.
100 ml demineralized water with constant stirring. Cloxacillin and
liquid paraffin were added to sodium alginate solution. The final 2.8. Surface morphology analysis
mixture of sodium alginate aqueous solution and liquid paraffin
containing cloxacillin was homogenized for 10 min at 1000 rpm For the morphological study, beads were mounted on metal
using a homogenizer (BL 232, BIO-LAB Instruments Mfg. Co., India) grids using double-sided tape coated with gold under a vac-
and stirring at 4000 rpm continuously for 30 min until the stable uum. Surface morphologies of the beads were investigated with a
emulsion is formed. This resultant emulsion was dropped through Scanning Electron Microscope (SEM) (JSM-5310LV Scanning Micro-
23G needle into 5% (w/v) calcium chloride solution (100 ml) scope, Japan) at 15 kV.
and the added droplets were retained in the calcium chloride
solution for 15 min to complete the curing reaction to produce 2.9. Fourier transform infrared (FTIR) spectroscopy analysis
spherical beads. Then the prepared cloxacillin alginate beads were
filtered and washed twice with petroleum ether and kept the Samples were reduced to powder and analyzed as KBr pellets
beads after drying at room temperature for 24 h. The dried liquid by using a Fourier transform infrared (FTIR) spectroscope (Perkin
paraffin-entrapped calcium alginate beads containing cloxacillin Elmer Spectrum RX I, USA). The pellet was placed in the sample
were stored in a desiccator until used. holder and spectral scanning was taken in the wavelength region
between 4000 and 600 cm−1 at a resolution of 4 cm−1 with scan
2.4. Determination of drug entrapment efficiency speed of 1 cm/s.

An accurately weighed amount of 100 mg of liquid paraffin- 2.10. In vitro drug release studies
entrapped calcium alginate beads containing cloxacillin were
placed into 100 ml of 0.1 N HCl (pH 1.2) with constant stirring The in vitro release of the drug (cloxacillin) from various liq-
for 6 h at 4000 rpm by magnetic stirrer (Remi Motors, India) uid paraffin-entrapped calcium alginate beads was tested using
at 37 ± 0.5 ◦ C and the resulting solution was filtered through a dissolution apparatus USP/BP/IP (Campbell Electronics, India).
Whatman® filter paper (No. 40). The drug content in the fil- An equivalent weight of beads containing 100 mg cloxacillin was
trate was determined spectrophotometrically using a UV–visible placed into 900 ml of in simulated gastric fluid (pH 1.2) maintained
spectrophotometer (Thermo Spectronic UV-1, USA) by measuring at 37 ± 0.5 ◦ C and stirring at 50 rpm. 10 ml of aliquots was collected
absorbance at max , 344 nm. The drug entrapment efficiency of at regular time intervals, and the same amount of fresh dissolu-
beads was calculated by using this following formula: tion medium was replaced into dissolution vessel to maintain the
sink condition throughout the experiment. The collected aliquots
Drug encapsulation efficeincy (%) were filtered, and further diluted suitably to analyze for the spec-
actual drug content in beads trophotometrically using UV–visible spectrophotometer (Thermo
= × 100
theoretical drug content in beads Spectronic UV-1, USA) at 344 nm.

2.5. Determination of bead size 2.11. Analysis of in vitro drug release kinetics and mechanism

The diameters of dried beads were measured using digital slide In order to predict and correlate the release behaviour of
calipers (Digmatic Masschieber, CD-6′′ CS, Mitutoyo Corporation, cloxacillin in simulated gastric fluid (pH 1.2) from these liquid
Japan) by inserting the beads in between the space of two metallic paraffin-entrapped calcium alginate beads, it is necessary to fit
plate and diameter of resultant beads were displayed in the digital into a suitable mathematical model. The in vitro drug release data
screen of the previously calibrated equipment. The average size was from various TSP-alginate composite beads containing diclofenac
then calculated by measuring the diameter of 3 sets of 20 beads sodium were evaluated kinetically various mathematical models
from each batch. like zero order, first order, Higuchi, and Korsmeyer–Peppas model
[24–27].
2.6. Density measurement Zero-order model: F = K0 t; where, F represents the fraction of
drug released in time t, and K0 is the apparent release rate constant
The mean weights and diameters of the beads were measured or zero-order release constant.
and used to calculate the densities of these spherical liquid paraffin- First-order model: ln(1 − F) = −K1 st t; where, F represents the
entrapped calcium alginate beads containing cloxacillin using the fraction of drug released in time t, and K1 is the first-order release
following equations: constant.
Higuchi Model: F = KH t1/2 ; where, F represents the fraction of
M 4
D= , and V = r 3 drug released in time t, and KH is the Higuchi dissolution constant.
V 3 Korsmeyer–Peppas Model: F = KP tn ; where, F represents the
where D is the density of the beads; M is the weight of the beads; fraction of drug released in time t, KP is the rate constant and n
V is the volume of the beads; r is the radius of the beads. is the release exponent, indicative of the drug release mechanism.
Again, the Korsmeyer–Peppas model was employed in the in
2.7. In vitro buoyancy study vitro drug release behaviour analysis of these formulations to dis-
tinguish between competing release mechanisms: Fickian release
The buoyancy ability of beads was determined using USP type II (diffusion-controlled release), non-Fickian release (anomalous
(Paddle type) dissolution apparatus (Campbell Electronics, India). transport), and case-II transport (relaxation-controlled release). For
50 beads were placed in the dissolution vessel containing 500 ml spheres, a value of n ≤ 0.43 indicates the Fickian release. The n value
 J. Malakar et al. / International Journal of Biological Macromolecules 50 (2012) 138–147 141

Fig. 2. The Fourier transform-infrared (FTIR) spectra of sodium alginate (a), liquid
paraffin-entrapped calcium alginate blank beads (b), pure cloxacillin (c) and liquid
paraffin-entrapped calcium alginate beads containing cloxacillin, F-O (d).

Fig. 1. Scanning electron microphotograph of liquid paraffin-entrapped calcium

paraffin-entrapped calcium alginate beads containing cloxacillin
alginate bead containing cloxacillin showing rough surface with pores and channels
(F-O). prepared by emulsion–gelation technique had significant charac-
ters of cloxacillin in the FTIR spectra, suggesting, there were no
reactions between the cloxacillin and the other excipients used.
between 0.43 and 0.85 is an indication of non-Fickian release (both
diffusion controlled and swelling controlled drug release). When,
n ≥ 0.85, it is case-II transport and this involves polymer dissolution 3.3. Optimization
and polymeric chain enlargement or relaxation [28,29].
Pharmaceutical scientists often face the challenge of finding the
appropriate combination of variables that will produce the product
2.12. Statistical analysis
with optimum properties. Traditionally, pharmaceutical formula-
tors develop formulations by changing one variable at a time and
All measured data are expressed as mean ± standard deviation
the method is time consuming. It is therefore important to under-
(S.D.). Each measurement was done in triplicate (n = 3).
stand the influence of formulation variables on the formulation
quality with a minimal number of experimental trials and sub-
3. Results and discussion sequent selection of formulation variables to develop optimized
formulation using established statistical tools such as factorial
3.1. Surface morphology analysis design [31]. The optimization technique encompasses designing a
set of experiment that will reliably measure the response variables,
Scanning electron microphotograph of liquid paraffin- fitting a mathematical model to the data, conducting appropriate
entrapped calcium alginate bead surface showed a rough surface statistical test to assure that the best possible model is chosen,
with small pores or channels (Fig. 1). The surface had an “orange and determining the optimum value of independent variables that
peel” appearance with corrugations. No large crystals of drug were produce best response [32].
found on the surface of these beads, indicating the presence of For the 32 factorial design, a total 9 experimental trial formu-
drug particles as finely dispersed state in the alginate matrix. lations were proposed by Design-Expert 8.0.3 software (Stat-Ease
Inc., USA) for two factors, drug to polymer ratio by weight (X1 ) and
3.2. FTIR analysis oil to water ratio by volume (X2 ), which were varied at three differ-
ent levels (high, medium and low). According to this experimental
The FTIR spectra of sodium alginate, liquid paraffin-entrapped trial proposal, various liquid paraffin-entrapped calcium alginate
calcium alginate blank beads, pure cloxacillin and liquid paraffin- beads containing cloxacillin were prepared by emulsion–gelation
entrapped calcium alginate beads containing cloxacillin were method. The effects of independent variables (factors) upon the
analyzed (Fig. 2). FTIR spectra of sodium alginate (Fig. 2a) showed drug entrapment efficiency (%), density (g/cm3 ), and drug release
the band around 3445, 1614, 1417 and 1032 cm−1 , which are due after 8 h (%) in simulated gastric fluid (pH 1.2) were investigated
to the stretching of –OH, –COO (asymmetric), –COO (symmetric), as optimization response parameters in this study. Overview of
and C–O–C, respectively. In the FTIR spectra of liquid paraffin- the experimental plan and observed responses were presented in
entrapped calcium alginate blank beads (Fig. 2b) showed only a Table 1.
shift of –COO stretching bands to higher wave number, indicating Suitable polynomial equations involving individual main fac-
the formation of ionic bonding between Ca2+ and –COO of sodium tors and interaction factors were selected based on the estimation
alginate due to cross-linking [30]. On the other hand, the princi- of several statistical parameters, such as multiple correlation coef-
pal FTIR peaks of pure cloxacillin (Fig. 2c) were observed at 3466, ficient (R2 ), and the predicted residual sum of squares (PRESS),
1659, 1510 and 1335 cm−1 , which are due to the stretching of –OH, provided by the Design-Expert 8.0.3 software. As presented in
–C O, –C C– and –COO, respectively. In the FTIR spectra of liquid Table 2, three quadratic models were selected as suitable statistical
paraffin-entrapped calcium alginate beads containing cloxacillin models for optimization of drug entrapment efficiency (%), density
(Fig. 2d), various characteristic peaks of liquid paraffin-entrapped (g/cm3 ) and drug release after 8 h (%) in simulated gastric fluid (pH
calcium alginate blank beads, and pure cloxacillin were appeared 1.2), because these models had smaller values of PRESS. PRESS is
without any significant shifting of these peaks. In short, the liquid measure of the fit of the model to data points in the design. The
142 J. Malakar et al. / International Journal of Biological Macromolecules 50 (2012) 138–147

Table 2
Summary of results of: (A) model analysis, (B) R2 analysis for measured responses.

Sources Drug entrapment efficiency (%) Density (g/cm3 ) Drug release after 8 h (%)

Sum of squares p-Value Sum of squares p-Value Sum of squares p-Value

(A) Model analysis

Mean vs Total 35201.26 6.90 13769.46
Linear vs Mean 60.08 0.0007 0.08 0.0008 123.69 0.0009
2FIa vs Linear 3.76 0.0272 0.00 0.3935 3.84 0.2129
Quadratic vs 2FIa 1.78 0.0304 0.01 0.0055 8.66 0.0235
Cubic vs Quadratic 0.08 0.7751 0.00 0.7876 0.59 0.4811
Residual 0.12 0.00 0.18
Total 35267.08 6.99 13906.42

Sources Drug entrapment efficiency (%) Density (g/cm3 ) Drug release after 8 h (%)

R2 PRESS R2 PRESS R2 PRESS

2
(B) R analysis
Linear 0.9128 15.95 0.9079 0.02 0.9031 30.56
2FIa 0.9700 3.96 0.9216 0.02 0.9311 21.18
Quadratic 0.9971 1.80 0.9976 0.00 0.9943 8.59
Cubic 0.9982 21.07 0.9985 0.03 0.9987 32.66
a
Two factor interaction.

smaller the PRESS statistic is, the better the model fits to the data The model equation relating drug released after 8 h (%) in sim-
points [22]. The results of the ANOVA indicated that these models ulated gastric fluid (pH 1.2) as response became:
were significant for all response parameters (Table 3).
The model equation relating drug entrapment efficiency (%) as Y3 = 58.58 + 92.17 X1 − 159.92 X2 − 78.40 X1 X2 − 60.69 X12
response became:
+ 185.17 X22
Y1 = 70.07 + 50.08 X1 − 44.70 X2 − 77.60 X1 X2 − 46.08 X12
From the ANOVA results (Table 3) of the model relating drug
+ 61.00X22 release after 8 h (%) in simulated gastric fluid (pH 1.2) as response,
it can be noted that only the coefficient, b4 had no statistic signif-
icance (p > 0.05) with the model F-value of 105.55 and R2 value of
From the ANOVA results (Table 3) of the model relating drug 0.9943.
entrapment efficiency (%) as response, it can be noticed that all Model simplification was carried out by eliminating non-
the coefficients of this model equation had statistic significance significant terms (p > 0.05) in the polynomial equation resulting
(p < 0.05) with the model F-value of 204.66 and R2 value of 0.9971. from the multiple regression analysis [33], giving:
The model equation relating density (g/cm3 ) as response
became: Y1 = 70.07 + 50.08 X1 − 44.70 X2 − 77.60 X1 X2 − 46.08 X12

Y2 = 1.54 − 3.16 X1 + 0.48 X2 − 1.40 X1 X2 + 3.73 X12 − 0.17 X22 + 61.00 X22
It can be noted from the ANOVA results (Table 3) of the model
relating density (g/cm3 ) as response that only the coefficient, b5 had Y2 = 1.54 − 3.16X1 + 0.48X2 − 1.40X1 X2 + 3.73X12
no statistic significance (p > 0.05) with the model F-value of 244.44
and R2 value of 0.9976. Y3 = 58.58 + 92.17 X1 − 159.92 X2 − 78.40 X1 X2 + 185.17 X22

Table 3
Summary of ANOVA for the response parameters.

Source Sum of squares d.f.a Mean square F value p-ValueProb > F

(a) For drug entappment efficiency (%)

Model 65.63 5 13.13 204.66 0.0005 (S)
X1 22.58 1 22.58 352.10 0.0003 (S)
X1 X2 3.76 1 3.76 58.68 0.0046 (S)
X12 1.04 1 1.04 16.17 0.0276 (S)
X22 0.74 1 0.74 11.60 0.0423 (S)
(b) For density
Model 0.09 5 0.02 244.44 0.0004 (S)
X1 0.08 1 0.08 1084.78 <0.0001 (S)
X2 0.00 1 0.00 27.57 0.0135 (S)
X1 X2 0.00 1 0.00 16.75 0.0264 (S)
X12 0.01 1 0.01 93.04 0.0024 (S)
X22 0.00 1 0.00 0.08 0.8008 (NS)
(c) For drug release after 8 h
Model 136.19 5 27.24 105.55 0.0014 (S)
X1 21.93 1 21.93 84.97 0.0027 (S)
X2 101.76 1 101.76 394.34 0.0003 (S)
X1 X2 3.84 1 3.84 14.89 0.0308 (S)
X12 1.80 1 1.80 6.97 0.0777 (NS)
X22 6.86 1 6.86 26.57 0.0142 (S)

X1 and X2 represent the main effects (factors); X12 and X22 are the quadratic effect; X1 X2 is the interaction effect. S and NS indicate significant and not significant, respectively.
a
Degree of freedom.
 J. Malakar et al. / International Journal of Biological Macromolecules 50 (2012) 138–147 143

Table 4
Comparison of actual and predicted responses for all response variables with parentage of errors in prognosis.

Batch code Responses Actual valuea Predicted value % Errorb

Drug entrapment efficiency (%) 66.11 ± 0.42 65.97 0.21

F-1 Density (g/cm3 ) 1.01 ± 0.04 1.01 0.00
Drug release after 8 h (%) 40.26 ± 0.64 40.04 −0.94
Drug entrapment efficiency (%) 62.89 ± 0.74 62.91 −0.03
F-2 Density (g/cm3 ) 1.01 ± 0.05 1.01 0.00
Drug release after 8 h (%) 34.34 ± 0.70 34.37 −0.09
Drug entrapment efficiency (%) 63.72 ± 0.83 63.83 −0.17
F-3 Density (g/cm3 ) 1.01 ± 0.04 1.01 0.00
Drug release after 8 h (%) 36.06 ± 0.38 35.65 1.07
Drug entrapment efficiency (%) 60.77 ± 0.57 60.72 0.08
F-4 Density (g/cm3 ) 0.82 ± 0.03 0.82 0.00
Drug release after 8 h (%) 36.03 ± 0.42 36.25 −0.61
Drug entrapment efficiency (%) 59.84 ± 0.43 59.95 −0.18
F-5 Density (g/cm3 ) 0.79 ± 0.02 0.78 1.28
Drug release after 8 h (%) 39.35 ± 0.75 39.48 −0.33
Drug entrapment efficiency (%) 62.84 ± 0.84 62.61 0.37
F-6 Density (g/cm3 ) 0.83 ± 0.03 0.84 −1.19
Drug release after 8 h (%) 38.23 ± 0.28 38.51 −0.73
Drug entrapment efficiency (%) 57.07 ± 0.76 57.09 −0.04
F-7 Density (g/cm3 ) 0.74 ± 0.01 0.74 0.00
Drug release after 8 h (%) 36.47 ± 0.64 36.23 0.66
Drug entrapment efficiency (%) 64.17 ± 0.66 64.03 0.22
F-8 Density (g/cm3 ) 0.81 ± 0.02 0.82 −1.22
Drug release after 8 h (%) 46.31 ± 0.60 46.43 −0.26
Drug entrapment efficiency (%) 65.45 ± 0.58 65.72 −0.41
F-9 Density (g/cm3 ) 0.86 ± 0.03 0.85 1.18
Drug release after 8 h (%) 44.98 ± 0.57 44.48 1.12
a
Mean ± S.D.; n = 3.
b
Percentage of error (%) = (actual value − predicted value)/predicted value × 100.

For all these 9 trial formulations proposed by the 32 factorial The influences of main effects (factors) on responses (here, drug
designs, the results of investigated responses (drug entrapment entrapment efficiency, density and drug released after 8 h) were
efficiency, density, and drug released at 8 h in simulated gastric further elucidated by response surface methodology. Response
fluid) were found within limits. Table 4 lists the actual and pre- surface methodology is a widely proficient approach in the
dicted responses for all response variables investigated in this study development and optimization of drug delivery devices [22,23].
with parentage of errors in prognosis. Percentage error evaluation The three-dimensional response surface graphs (Figs. 9–11) and
is helpful in establishing the validity of generated model equations the Design-Expert 8.0.3 software generated corresponding two-
and also to describe the domain of applicability of optimization dimensional contour plots (Figs. 12–14). The three-dimensional
model. Upon comparison of percentage error for all measured response surface graph is very useful in learning about the main and
responses were varied −1.22 to 1.28%. Linear correlation plots interaction effects of the independent variables (factors), whereas
between the actual, the predicted response variables are presented two-dimensional contour plot gives a visual representation of
in Figs. 3–5, and their corresponding residual plots showing the values of the response [31]. The three-dimensional response sur-
scatter of the residuals versus predicted values are presented in face graph relating drug entrapment efficiency (Fig. 9) depicts the
Figs. 6–8. increase in drug entrapment with the decreasing of both drug to

Fig. 3. Linear correlation plot between the actual and the predicted values for drug Fig. 4. Linear correlation plot between the actual and the predicted values for den-
entrapment efficiency (%). sity (g/cm3 ).
144 J. Malakar et al. / International Journal of Biological Macromolecules 50 (2012) 138–147

Fig. 5. Linear correlation plot between the actual and the predicted values for drug Fig. 7. Residual plot showing the scatter of the residuals versus predicted values for
release after 8 h (%). density (g/cm3 ).

polymer ratio (X1 ), and liquid paraffin to water ratio (X2 ). On the
other hand, the three-dimensional response surface graph relat-
ing density (Fig. 10) indicates the decrease of density with the
increasing of both drug to polymer ratio (X1 ), and liquid paraffin to
water ratio (X2 ). But, increase in drug release from liquid paraffin-
entrapped calcium alginate beads containing cloxacillin after 8 h
with the increasing of drug to polymer ratio (X1 ), and decreasing
of liquid paraffin to water ratio (X2 ) is indicated by the three-
dimensional response surface graph relating drug release (Fig. 11).
All the two-dimensional contour plots relating measured responses
(Figs. 12–14) were found to be nonlinear, which indicate nonlin-
ear relationships between two independable variables (here, drug
to polymer ratio, X1 and liquid paraffin to water ratio, X2 ) on all
measured responses, investigated in this study.
A numerical optimization technique using the desirability
approach was employed to develop new formulations with desired
response (desired quality). The desirable ranges of the inde-
pendable variables (factors) were restricted to 0.40 ≤ X1 ≤ 0.60,
and 0.10 ≤ X2 ≤ 0.25; whereas the desirable ranges of responses
Fig. 8. Residual plot showing the scatter of the residuals versus predicted values for
drug release after 8 h (%).

Fig. 9. Effect of drug to polymer ratio and liquid paraffin to water ratio on drug
Fig. 6. Residual plot showing the scatter of the residuals versus predicted values for
entrapment efficiency (%) presented by response surface plot.
drug entrapment efficiency (%).
 J. Malakar et al. / International Journal of Biological Macromolecules 50 (2012) 138–147 145

Table 5
Results of experiments for confirming optimization capability.

Batch code Drug to polymer Oil to water ratio Drug entrapment Density (g/cm3 )a Drug release after 8 h (%)a
ratio (by weight) (by volume) efficiency (%)a

Actual values
F-O 0.58 0.22 64.63 ± 0.78 0.90 ± 0.05 56.72 ± 0.85
Predicted values
F-O 0.58 0.22 66.53 0.88 54.86
% Errorb −2.86 2.27 3.39
a
Mean ± S.D.; n = 3.
b
Percentage of error (%) = (actual value − predicted value)/predicted value × 100.

were restricted to 65% ≤ Y1 ≤ 70%, 0.88 ≤ Y2 ≤ 0.92 g/cm3 , and (%) in simulated gastric fluid (pH 1.2). Table 5 lists the results
40% ≤ Y3 ≤ 55%. The optimal values of responses were obtained by of experiments with predicted responses by the mathematical
numerical analysis using the Design-Expert 8.0.3 software based model and those actually observed. The optimized liquid paraffin-
on the criterion of desirability. In order to evaluate optimiza- entrapped calcium alginate beads containing cloxacillin (F-O)
tion capability of models generated according to the results of showed drug entrapment efficiency of 64.63 ± 0.78%, density of
the full 32 factorial design, optimized liquid paraffin-entrapped 0.90 ± 0.05 g/cm3 , and drug release of 56.72 ± 0.85% in simulated
calcium alginate beads containing cloxacillin was prepared by gastric fluid (pH 1.2) after 8 h with small error-values (−2.86, 2.27
emulsion–gelation technique using the optimal process variable
settings. The optimized liquid paraffin-entrapped calcium alginate
beads containing cloxacillin (F-O) was evaluated for drug entrap-
ment efficiency (%), density (g/cm3 ) and drug release after 8 h

Fig. 12. Effect of drug to polymer ratio and liquid paraffin to water ratio on drug
entrapment efficiency (%) presented by contour plot.
Fig. 10. Effect of drug to polymer ratio and liquid paraffin to water ratio on density
(g/cm3 ) presented by response surface plot.

Fig. 11. Effect of drug to polymer ratio and liquid paraffin to water ratio on drug Fig. 13. Effect of drug to polymer ratio and liquid paraffin to water ratio on density
release after 8 h (%) presented by response surface plot. (g/cm3 ) presented by contour plot.
146 J. Malakar et al. / International Journal of Biological Macromolecules 50 (2012) 138–147

Table 6
Bead size, buoyancy and buoyancy lag time of various dried liquid paraffin-
entrapped calcium alginate beads containing cloxacillin.

Batch code Diameter (mm)a Buoyancy (h) Buoyancy lag time (min)

F-1 1.93 ± 0.08 Sink –

F-2 1.96 ± 0.06 Sink –
F-3 1.95 ± 0.08 Sink –
F-4 1.84 ± 0.05 >12 8.10
F-5 1.82 ± 0.02 >12 8.00
F-6 1.84 ± 0.06 >12 8.20
F-7 1.77 ± 0.01 >12 7.30
F-8 1.75 ± 0.04 >12 8.15
F-9 1.73 ± 0.07 >12 8.30
F-O 1.85 ± 0.06 >12 8.45
a
Mean ± S.D.; n = 3.

analysis, we have found that the decreasing density values of

various dried liquid paraffin-entrapped calcium alginate beads con-
taining cloxacillin with the increasing of both drug to polymer
ratio, and liquid paraffin to water ratio. Results of lower densities
obtained in the presence of higher oil concentrations entrapped
in polymeric systems are in line with results reported in previous
Fig. 14. Effect of drug to polymer ratio and liquid paraffin to water ratio on drug
literature [16,19,20].
release after 8 h (%) presented by contour plot.

3.7. In vitro buoyancy lag time and buoyancy

and 3.39, respectively). This reveals that mathematical models
obtained from the full 32 factorial designs were well fitted. The in vitro buoyancy results of various dried liquid paraffin-
entrapped calcium alginate beads containing cloxacillin in
3.4. Drug entrapment efficiency simulated gastric fluid (pH 1.2) are presented in Table 6. Beads of
F-1, F-2, and F-3, which had the density more than 1 were failed
The drug entrapment efficiency of formulated liquid paraffin- to float (buoyant) in the tested medium. But, the density values
entrapped calcium alginate beads ranged from 57.07 ± 0.76 to of all other formulated beads (F-4 to F-9, and F-O) were less than
66.11 ± 0.42% (Tables 1 and 5) depending on the composition the density of in simulated gastric fluid (pH 1.2) (i.e., 1.004 g/cm3 )
of the 10 batches (9 trial batches and 1 optimized batch). The imparting their flotation (buoyancy) and these beads were floated
drug entrapment in the liquid paraffin-entrapped calcium alginate well over 12 h. These beads were floated within 10 min after being
containing cloxacillin was found to be increased with higher pro- placed in the acidic medium (in simulated gastric fluid, pH 1.2). The
portion liquid paraffin in these beads. This phenomenon may be entrapped oil (here liquid paraffin) in the beads was responsible for
due to either partitioning of some amount of drug (cloxacillin) in floating. Actually, entrapped low-density oils can lower the density
the oil (liquid paraffin) phase or formation of oil barrier, which of the polymeric systems with incorporation. Thus, a clear corre-
obstructed the passage of drug molecules to the external media lation was evidenced between the density values of these liquid
during preparation. Moreover, this was seen that an increase in the paraffin-entrapped calcium alginate beads containing cloxacillin
proportion of sodium alginate in the bead formulations increased with their buoyancy and buoyancy lag time.
the drug entrapment efficiency also due to physical interaction
and/or entanglement of higher amount of drug inside intricate 3.8. In vitro drug release
cross-linked calcium alginate gel network.
The in vitro drug release studies were carried out for all
3.5. Bead size formulated liquid paraffin-entrapped calcium alginate beads con-
taining cloxacillin in simulated gastric fluid (pH 1.2). Various
The size of dried liquid paraffin-entrapped calcium alginate beads (F-1 to F-9, and F-O) showed prolonged sustained release of
beads containing cloxacillin for each formulation was measured cloxacillin over 8 h (Fig. 15). An increase in drug release from liquid
using digital slide calipers (Digmatic Masschieber, CD-6′′ CS, Mitu- paraffin-entrapped calcium alginate beads containing cloxacillin
toyo Corporation, Japan). The average size of these dried beads was observed with the increasing of polymer content and decreas-
ranged from 1.73 ± 0.04 to 1.97 ± 0.08 mm (Table 6). Increasing the ing of liquid paraffin content in beads. The most reasonable
bead size was found with the increasing proportion sodium algi- explanation of the slow sustained release was that the most of the
nate into formulations. This could be attributed due to the increase drugs remained saturated and dispersed in the oil pockets of the
in viscosity of emulsion with incorporation sodium alginate in beads to form a drug-oil dispersed matrix. Actually, drug trans-
increasing ratio that in turn increased the droplet size during addi- portation from beads to the dissolution medium may undergo two
tion of the liquid paraffin-entrapped sodium alginate-cloxacillin steps [16]. Firstly, the drug may diffuse out of oil pockets into cal-
containing emulsion to the cross-linking solution. cium alginate matrix. Secondly, it may diffuse out of the calcium
alginate matrix in to the dissolution medium.
3.6. Density The in vitro drug release data from various liquid paraffin-
entrapped calcium alginate beads containing cloxacillin were
Density values of various dried liquid paraffin-entrapped cal- evaluated kinetically using various mathematical models like
cium alginate beads containing cloxacillin ranged from 0.74 ± 0.02 zero order, first order, Higuchi, and Korsmeyer–Peppas model.
to 1.01 ± 0.05 g/cm3 (Tables 1 and 5). Among all these beads, The results of the curve fitting into these above-mentioned
only F-1, F-2, and F-3 beads had density more than 1 and other mathematical models are given in Table 7. When respective cor-
beads showed the density less than 1. From the response surface relation coefficients of these beads in simulated gastric fluid were
 J. Malakar et al. / International Journal of Biological Macromolecules 50 (2012) 138–147 147

cloxacillin. The ionotropically emulsion–gelation method for the

preparation of these liquid paraffin-entrapped calcium alginate
floating beads containing cloxacillin system for gastroretentive
delivery was found to be simple, reproducible, easily controllable,
economical and consistent process. Additionally, the excipients
used for the formulation of these multiple-unit buoyant systems
were cheap and easily available.

Acknowledgement

One of the authors is thankful to Dr. R.M. Dubey, Vice Chancel-

lor, IFTM University, Moradabad, 244 102, U.P., India for providing
computer facilities.

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