Gum ghatti–chitosan polyelectrolyte nanoparticles: Preparation and characterization

Gum ghatti–chitosan polyelectrolyte nanoparticles: Preparation and characterization

International Journal of Biological Macromolecules 61 (2013) 411–415 Contents lists available at ScienceDirect International Journal of Biological M...

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International Journal of Biological Macromolecules 61 (2013) 411–415

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac

Short communication

Gum ghatti–chitosan polyelectrolyte nanoparticles: Preparation and characterization Shelly, Munish Ahuja ∗ , Ashok Kumar Drug Delivery Research Laboratory, Department of Pharmaceutical Sciences, Guru Jambheshwar University of Science and Technology, Hisar 125 001, India

a r t i c l e

i n f o

Article history: Received 14 May 2013 Received in revised form 2 July 2013 Accepted 30 July 2013 Available online 4 August 2013 Keywords: Antibacterial Chitosan Gum ghatti Ofloxacin Polyelectrolyte nanoparticles

a b s t r a c t The objective of the present study was to optimize the interaction between gum ghatti and chitosan to prepare polyelectrolyte nanoparticles using ofloxacin as the model drug. The effect of varying the concentration of gum ghatti, chitosan, Pluronic F-127, and ofloxacin on particle size and entrapment efficiency was studied using central composite experimental design. The optimized calculated parameters were concentrations of gum ghatti (0.12% w/v), chitosan (0.22% w/v), Pluronic F-127 (0.05% w/v), ofloxacin (0.1% w/v), which provided polyelectrolyte nanoparticles of size 121.6 nm and 94.49% entrapment. On screening for antibacterial activity, it was observed that polyelectrolyte nanoparticles had antibacterial activity comparable to the aqueous solution. Further, it was observed that polyelectrolyte nanoparticles released the drug by diffusion through the matrix following Higuchi’s square-root kinetics. © 2013 Elsevier B.V. All rights reserved.

1. Introduction

2. Experimental

Gum ghatti, an anionic polysaccharide is obtained as exudate from Anogeissus latifolia (family Combretaceae). It has been granted the status of Generally Recognized as Safe (GRAS). It has been used widely as food additive and pharmaceutically as emulsifier, thickner and binder [1,2]. Gum ghatti reportedly comprises of arrays of neutral sugar units (Galp, Araf, and Arap) and GlcA attached to alternating ␤-d GlcA and d-Man residue [3]. It occurs naturally as Ca2+ , Mg2+ salt. It exhibits ion concentration and pH dependent viscosity. Its viscosity increases with Ca2+ ions and decreases with Na+ ions. It shows maximum viscosity in pH range of 6–8. During earlier study, the properties of gum ghatti were modified by grafting with acrylamide [4] and acrylonitrile [5] to prepare temperature, pH responsive, superabsorbent hydrogels etc. It has also been explored as sustained release matrix tablet by forming polyelectrolyte complex with chitosan [6]. In the present study, the interaction between gum ghatti and chitosan has been explored to prepare polyelectrolyte nanoparticles using ofloxacin as a model drug. The interaction between two electrolytes has been optimized using response surface methodology to achieve nanosuspension having minimum particle size and maximum entrapment efficiency. Further, polyelectrolyte nanoparticles were screened for antibacterial activity and in vitro release behavior.

2.1. Materials

∗ Corresponding author. Tel.: +91 1662 263515; fax: +91 1662 276240. E-mail address: [email protected] (M. Ahuja). 0141-8130/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijbiomac.2013.07.022

Gum ghatti was procured from Central Drug House Pvt. Ltd. (New Delhi, India). Chitosan (Degree of deacetylation-80%) was received as a gift sample from Sea Foods, (Cochin, India). Pluronic (PF-127) and ofloxacin were gifted by Ranbaxy Research Laboratories (Gurgaon, India). All other chemicals and reagents were used as received. 2.2. Preparation of gum ghatti–chitosan polyelectrolyte complex nanoparticles Nanoparticles of gum ghatti and chitosan were prepared employing the interaction between anionic gum ghatti and cationic chitosan to give polyelectrolyte complex employing ofloxacin as a model drug [7]. Briefly, an aqueous dispersion of chitosan in acetic acid was prepared with the aid of stirring. To this Pluronic F-127 and ofloxacin were added. An aqueous dispersion of gum ghatti was prepared in with the aid of heating. Gum ghatti dispersion was sprayed in chitosan solution using spray gun. 2.3. Experimental design The preparation of ofloxacin-loaded gum ghatti–chitosan polyelectrolyte nanoparticles was optimized using a central composite experimental design. Concentrations of gum ghatti, chitosan, Pluronic F-127 and ofloxacin were selected as independent

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Table 1 The results of response generated using the experimental design. Batch no.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

Concentration (%)

PS (Y1 )

GG (X1 )

CH (X2 )

PF-127 (X3 )

OFL (X4 )

0.10(−1) 0.50(+1) 0.10(−1) 0.50(+1) 0.10(−1) 0.50(+1) 0.10(−1) 0.50(+1) 0.10(−1) 0.50(+1) 0.10(−1) 0.50(+1) 0.10(−1) 0.50(+1) 0.10(−1) 0.50(+1) 0.10(−1) 0.50(+1) 0.30(0) 0.30(0) 0.30(0) 0.30(0) 0.30(0) 0.30(0) 0.30(0) 0.30(0) 0.30(0) 0.30(0) 0.30(0) 0.30(0)

0.05(−1) 0.05(−1) 0.25(+1) 0.25(+1) 0.05(−1) 0.05(−1) 0.25(+1) 0.25(+1) 0.05(−1) 0.05(−1) 0.25(+1) 0.25(+1) 0.05(−1) 0.05(−1) 0.25(+1) 0.25(+1) 0.15(0) 0.15(0) 0.05(0) 0.25(+1) 0.15(0) 0.15(0) 0.15(0) 0.15(0) 0.15(0) 0.15(0) 0.15(0) 0.15(0) 0.15(0) 0.15(0)

0.05(−1) 0.05(−1) 0.05(−1) 0.05(−1) 0.50(+1) 0.50(+1) 0.50(+1) 0.50(+1) 0.05(−1) 0.05(−1) 0.05(−1) 0.05(−1) 0.50(+1) 0.50(+1) 0.50(+1) 0.50(+1) 0.28(0) 0.28(0) 0.28(0) 0.28(0) 0.05(−1) 0.50(+1) 0.28(0) 0.28(0) 0.28(0) 0.28(0) 0.28(0) 0.28(0) 0.28(0) 0.28(0)

0.050(−1) 0.050(−1) 0.050(−1) 0.050(−1) 0.050(−1) 0.050(−1) 0.050(−1) 0.050(−1) 0.100(+1) 0.100(+1) 0.100(+1) 0.100(+1) 0.100(+1) 0.100(+1) 0.100(+1) 0.075(0) 0.075(0) 0.075(0) 0.075(0) 0.075(0) 0.075(0) 0.075(0) 0.050(−1) 0.100(+1) 0.075(0) 0.075(0) 0.075(0) 0.075(0) 0.075(0) 0.075(0)

nm 139.1 506.6 148.7 205.7 165.3 348.6 253.9 215.1 155.5 431.7 122 187.8 1817 548.9 257.5 191.7 251.5 390.4 429 158.8 147.7 159.3 147 220.6 204.1 187.3 203.4 194.8 156.9 193.1

EE (Y2 ) PdI

%

1 0.519 0.234 0.531 0.220 0.652 0.593 0.301 0.514 0.161 0.205 0.120 0.123 0.208 0.577 0.109 0.069 0.143 0.015 0.072 0.195 0.568 0.062 0.580 0.075 0.072 0.410 0.037 0.637 0.710

98.94 88 97.62 89.33 93.16 87.93 96.89 89.28 97.11 94.69 99.55 94.85 99.53 94.16 97.9 94.86 93.04 97.74 92.93 97.9 96.3 97.91 96.7 94.66 92.85 92.96 97.54 97.11 94.2 97.03

Value in parenthesis indicated coded values, GG: gum ghatti, CH: chitosan, PF-127: Pluronic, OFL: ofloxacin, PS: particle size, EE: encapsulation efficiency, PdI: polydispersity index.

variables on the basis of preliminary trials. Particle size and encapsulation efficacy were selected as dependent variables. The effect of varying the concentrations of gum ghatti, chitosan, Pluronic F127 and ofloxacin on the particle size and encapsulation efficiency was studied at three levels i.e., low (−1), medium (0), and high (+1) level as shown in Table 1. The experimental design and statistical analysis of the data were done using the Design Expert software (Version 7.1.6, Stat-Ease Inc., Minneapolis, MN).

3.3. Morphology

3. Characterization of nanoparticles

The optimized batch of ofloxacin-loaded gum ghatti–chitosan polyelectrolyte nanoparticles was evaluated comparatively with aqueous ofloxacin solution for antibacterial activity by agar plate diffusion method against the strains of Bacillus subtilis (MTCC 2063), Escherichia coli (MTCC 1652), Staphylococcus aureus (MTCC 2901), Micrococcus luteus (M-37) and Pseudomonas aeruginosa (PA-168) [9]. Agar plates were prepared aseptically from sterile nutrient agar and inoculated with 10 ␮l of 24 h broth cultures of microbial strains. A central cavity was made in the agar plate by means of sterile steel cork borer. A 10 ␮l of optimized batch of ofloxacin (0.1% w/v) loaded-gum ghatti–chitosan nanoparticles, or ofloxacin solution (0.1% w/v) or blank gum ghatti–chitosan nanoparticles were added to the central cavity. The agar plates were kept at room temperature for 4 h to allow for the diffusion of drug followed by incubation at 37 ◦ C for 24 h. The plates were observed for growth after incubation period and zone of inhibition was measured. All the experiments were done in triplicate.

3.1. Particle size Average particle size and size distribution (polydispersity index) was measured by dynamic light scattering using Zetasizer Nano ZS (Malvern Instruments, UK). 3.2. Encapsulation efficiency The encapsulation efficiency of ofloxacin-loaded nanoparticles was determined by separating the unentrapped drug from the nanoparticles by centrifugation at 11,000 rpm at 5 ◦ C for 45 min [Cooling centrifuge C-24BL, Remi Instruments, Mumbai]. The amount of ofloxacin entrapped in nanoparticles was calculated as the difference between the total ofloxacin (OFt ) used in nanoparticles and ofloxacin found in supernatant (OFf ) The amount of free ofloxacin in supernatant was determined spectrophotometrically by measuring the absorbance in UV–vis spectrophotometer at 293 nm. The encapsulation efficiency (EE) was calculated as follows [8]. EE(%) =

OFt − OFf × 100 OFt

(1)

The shape of optimized batch of ofloxacin-loaded gum ghatti–chitosan nanoparticles was examined by transmission electron microscopy (TEM; Hitachi H7500). The micrographs were taken at an accelerating voltage of 90 kV. 3.4. Antibacterial activity

3.5. In vitro release studies The in vitro release behavior of ofloxacin from optimized batch of ofloxacin loaded gum ghatti–chitosan nanoparticulate formulation was evaluated by dialysis sac method [8]. A 3 ml sample of nanosuspension placed in dialysis sac (cut off 10,000 kDa), was

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Fig. 1. (a–d) Response surface plots showing combined effect of concentration of (a) gum ghatti and chitosan, (b) gum ghatti and Pluronic F-127, (c) chitosan and ofloxacin, (d) Pluronic F-127 and ofloxacin.

tied to the paddle of USP-type II (TDT-08L, Electrolab, Mumbai India) dissolution rate apparatus. The release media comprised of 200 ml of phosphate buffer (pH 7.4) maintained at 37 ± 0.5 ◦ C and stirred at 25 rpm. At appropriate time interval, 3 ml of dissolution medium was withdrawn and replaced with equal volume of dissolution medium. To study the limiting effect of dialysis membrane, experiments with ofloxacin solution (0.1% w/v) in dialysis membrane were also conducted. The withdrawn samples were analyzed for the contents of ofloxacin spectrophotometrically by measuring absorbance at 293 nm. 4. Results and discussion Gum ghatti, an anionic gum interacts with chitosan to form polyelectrolyte complex which was earlier explored as matrix for sustained delivery of diltiazem as tablet delivery system [10]. Anionic polysaccharides such as alginate [11,12], pectin [13], xanthan gum [14], gum kondagogu [15] on mixing with cationic chitosan interact to form polyelectrolyte complex. The formation of polyelectrolyte complex depends upon number of factors such as concentration of electrolytes, ionic strength, pH and order of mixing etc. [16]. From preliminary studies, it was observed that varying the concentrations of gum ghatti, chitosan in the presence of varying concentration of Pluronic F-127 as stabilizer affects the loading of ofloxacin (a model drug) and particle size of the polyelectrolyte particles. Thus in present study, the concentration of gum ghatti (0.10–0.50%), chitosan (0.05–0.25%), Pluronic F-127(0.05–0.5%) and ofloxacin (0.05–0.1%) were selected for finding the optimized concentration to prepare polyelectrolyte particles having minimum particle size and maximum entrapment efficiency. Table 1 presents the results of response generated using the experimental design. The results of the various batches of gum ghatti–chitosan polyelectrolyte complex nanoparticles prepared using the experimental design were fitted into various polynomial models followed by ANOVA analysis to estimate the significance of model. It was observed that the response particle size (Y1 ) fitted best into the quadratic model after inverse transformation of data while response entrapment efficiency (Y2 ) fitted best into linear model. The polynomial models for response particle size (Y1 ) and

response encapsulation efficiency (Y2 ) are represented by Eq. (2) and (3) respectively as follows: 1 0.00521 − 0.00085X1 + 0.00097X2 − 0.00080X3 − 0.00037X4 Y1 + 0.00053X1 X2 + 0.00088X1 X3 + 0.00032X1 X4 − 0.00005X2 X3 + 0.00058X2 X4 − 0.00047X3 X4 − 0.00185X12 − 0.00081X22 − 0.00139X32 − 0.00054X42 Y2 = 95.09 − 2.38X1 + 0.65X2 − .027X3 + 1.64X4

(2)

(3)

It can be observed that response particle size (Y1 ) was affected significantly by concentration of gum ghatti (X1 ), chitosan (X2 ), Pluronic F-127 (X3 ), and interaction effects of gum ghatti and chitosan (X1 X2 ), gum ghatti and Pluronic F-127 (X1 X3 ), chitosan and ofloxacin (X2 X4 ), Pluronic F-127 and ofloxacin (X3 X4 ), while the response encapsulation efficiency (Y2 ) was affected significantly by cocncentration of gum ghatti (X1 ) and ofloxacin (X4 ). Further, ANOVA analysis of the polynomial models revealed that the developed response surface models were significant (P value < 0.05), adequate and without significant ‘lack of fit’ (P value > 0.05). Fig. 1(a) portrays the combined effect of gum ghatti and chitosan on particle size. It can be inferred from the plot that a curvilinear relationship exists between the concentration of gum ghatti and particle size. The effect of gum ghatti on particle size is more prominent than chitosan. Increasing concentration of gum ghatti results in increase in particle size which may be attributed to the aggregation of particles at higher concentration. Fig. 1(b) displays the combined effect of gum ghatti and Pluronic F-127 on particle size and shows curvilinear relationship with one factor trying to modify the effect of another factor. It can be inferred from the plot that at low concentration of gum ghatti, increasing the concentration of Pluronic F-127 increase the particle size which may be attributed to its inhibition of interaction between gum ghatti and chitosan while at higher concentration of gum ghatti, this effect of Pluronic F-127 on particle size is attenuated. Fig. 1(c) represents the combined effect of chitosan and ofloxacin on particle size reveals that increasing the concentration of chitosan results in decrease

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Fig. 2. Response surface plot showing combined effect of concentration of gum ghatti and chitosan on encapsulation efficiency of ofloxacin.

in particle size while the concentration of ofloxacin has no significant effect on particle size. Fig. 1(d) shows the combined effect of Pluronic F-127 and ofloxacin on particle size. It can be observed that concentration of Pluronic F-12 has more prominent effect on particle size. Increasing the concentration of Pluronic F-127 decreases the particle size. Fig. 2 exhibits the combined effect of concentration of gum ghatti and chitosan on encapsulation efficiency of ofloxacin. It can be observed that increasing the concentration of gum ghatti decreases the encapsulation efficiency. Increase in concentration of gum ghatti increases its viscosity which results in increase in size of gum ghatti droplets being sprayed through spray gun resulting in inadequate interaction between chitosan and gum ghatti. As a result ofloxacin leaches out from the poorly gelled matrix leading to decrease in % encapsulation. On the other hand, increasing the concentration of chitosan results in increase in the amount of drug entrapped by providing enough concentration of chitosan for interaction with gum ghatti. To develop ofloxacin loaded-gum ghatti–chitosan polyelectrolyte complex nanoparticulate formulation with desired parameters, numerical optimization tool along with desirability approach was employed. The optimization was done with constraints for particle size to be minimum and maximum encapsulation efficiency as the goals to locate the optimum values of independent variables. The Design Expert software provided us with 55 solutions; out of this one with highest desirability was selected for preparing optimized formulation. The optimal calculated parameters were concentrations of gum ghatti (0.12%, w/v), chitosan (0.22%, w/v), Pluronic F-127 (0.05%) and ofloxacin (0.1%). To validate developed mathematical models, the response of optimal formulation of ofloxacin-loaded gum ghatti–chitosan polyelectrolyte nanoparticles and three additional checkpoints formulations in experimental domain were recorded. The results of particle size and entrapment efficiency as determined experimentally for these test runs were compared with the results predicted by polynomial models. Table 2 details the test conditions of optimal formulation and their experimental and predicted values along with calculated % error. The lower values of % prediction

Fig. 3. The transmission electron micrograph ghatti–chitosan polyelectrolyte nanoparticles.

of

ofloxacin

loaded-gum

error for particle size (Y1 ) (−0.26 to 11.59) and for entrapment efficiency (Y2 ) (−5.35 to 5.17) indicate robustness of developed mathematical models and high prognostic ability of response surface methodology. The transmission electron micrograph (Fig. 3) of ofloxacinloaded gum ghatti–chitosan polyelectrolyte complex nanoparticle shows ovoid shape aggregates with size of approximately 100 nm. The optimized batch of ofloxacin-loaded gum ghatti chitosan polyelectrolyte complex nanoparticles was further screened for antibacterial activity in comparison to ofloxacin solution. The results of antibacterial activity (Table 3) as measured by measuring zone of inhibition revealed that there was no significant difference (P > 0.05) in inhibition of growth produced by ofloxacin loaded nanoparticulate formulation as compared to aqueous solution of ofloxacin. Thus even though 94.49% of ofloxacin is entrapped in nanoparticulate formulation, the antibacterial activity is not compromised. Fig. 4 displays in vitro profile of ofloxacin from the optimized batch of gum-ghatti–chitosan polyelectrolyte complex nanoparticulate formulation. It can be inferred from the plot that the nanoparticulate formulations provided a sustained release of the drug with 27% of the drug getting released in 12 h. On fitting of release rate data into various kinetic models, the value of R2 for zero-order, first-order, Higuchi’s square-root kinetics and Korsemeyer–Peppas models was found to be 0.728, 0.759, 0.910 and 0.885 respectively. Further, the value of ‘n’ the release exponent of Korsemeyer–Peppas model was found to be 0.07 (n < 0.43) [17]. The results indicate that the nanoparticulate formulation released the drug following Higuchi’s square-root release kinetics with the mechanism of release being diffusion through the matrix.

Table 2 The test conditions of optimal and checkpoint formulations and their experimental and predicted values for response Y1 and Y2 . Checkpoints conditions

a

1 2 3 4 a

Particle size (nm) Y1

Encapsulation efficiency (%) Y2

X1

X2

X3

X4

Exp. value

Predi. value

% error

Exp. value

Predi. value

% error

0.12 0.15 0.17 0.14

0.22 0.24 0.25 0.25

0.05 0.05 0.07 0.06

0.10 0.09 0.10 0.10

121.6 124.3 138.0 135.9

121.92 122 122 122

−0.26 1.85 11.59 10.22

94.49 94.21 99.71 94.49

99.55 99.10 94.55 99.5

−5.35 −5.19 5.17 −5.30

Optimized batch.

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Table 3 Antibacterial activity of ofloxacin (0.1%, w/v) polyelectrolyte nanoparticles and ofloxacin solution (0.1%, w/v). Formulation

OGCPN OS (0.1% w/v) Polyelectrolyte nanoparticles

Zone of inhibition (mm)a Bacillus subtilis (MTCC 2063)

Staphylococcus aureus (MTCC 2901)

Escherichia coli (MTCC1652)

Pseudomonas aeruginosa (PA-168)

Micrococcus luteus (M-37)

26.00 ± 5.29 28.66 ± 3.05 0.00

26.00 ± 2.00 24.66 ± 3.05 0.00

29.00 ± 4.58 29.33 ± 1.52 0.00

32.00 ± 2.64 29.00 ± 1.00 0.00

25.33 ± 5.03 25.33 ± 0.57 0.00

OGCPN: ofloxacin loaded gum ghatti–chitosan polyelectrolyte nanoparticles, OS: ofloxacin solution. a Values are mean ± SD (n = 3).

Acknowledgement The authors are grateful to the University Grants Commission (UGC), New Delhi for providing financial assistance under the scheme of Major Research Project. References

Fig. 4. In vitro release profile of ofloxacin from ofloxacin loaded-gum ghatti–chitosan polyelectrolyte nanoparticles (OGCPN) and ofloxacin solution (OS).

5. Conclusion Interaction between gum ghatti and chitosan was optimized using response surface methodology to prepare nanosuspension using ofloxacin as the model drug. Entrapment of ofloxacin in the nanosuspension did not affect its anti-bacterial activity. The nanosuspension provided sustained release of ofloxacin. By varying the proportions of drug and polymer one can achieve the desired release rate. In conclusion gum ghatti–chitosan polyelectrolyte complex provide suitable means for sustained nanoparticulate delivery of drugs.

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