International Journal of Biological Macromolecules 73 (2015) 138–145
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Optimization, characterization and evaluation of chitosan-tailored cubic nanoparticles of clotrimazole Purnima Verma, Munish Ahuja ∗ Drug Delivery Research Laboratory, Department of Pharmaceutical Sciences, Guru Jambheshwar University of Science and Technology, Delhi-Bye pass Road, Hisar 125 001, Haryana, India
a r t i c l e
i n f o
Article history: Received 27 August 2014 Received in revised form 18 October 2014 Accepted 30 October 2014 Available online 24 November 2014 Keywords: Cubic nanoparticles Chitosan Central composite design
a b s t r a c t The present study deals with improvement of the mucoadhesive properties of monoolein based cubic nanoparticles by incorporating chitosan. Chitosan-tailored cubic nanoparticles were prepared by thin film hydration followed by ultrasonication employing clotrimazole as model drug. The effect of Pluronic F127 fraction and concentration of chitosan on particle size and % mucin binding of the formulations was studied using 2-factor, 3-level, central composite experimental design. The concentration of chitosan was found to influence particle size and % mucin binding of cubic nanoparticles while Pluronic F127 fraction influenced only the % mucin binding. Studies indicated 8.33(%w/w) fraction of Pluronic F127 and 0.17 (%w/v) concentration of chitosan as optimum concentration. Finally, the optimized batch was characterized by polarized light microscopy, small-angle X-ray scattering (SAXS) and transmission electron microscopy. The results unveiled incorporation of chitosan did not disrupt the inner cubic structure of nanoparticles. Peak indexing of SAXS data revealed the coexistence of P-type and D-type cubic phases in nanoparticles. Further, comparative evaluation studies showed significantly higher anti-fungal activity of clotrimazole-loaded chitosan-tailored cubic nanoparticles than conventional suspension of clotrimazole against Candida albicans. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Monoolein is a polar unsaturated long chain monoglyceride. It can self-associate in water to form thermodynamically stable, well defined cubic phases. The cubic phases which comprises of hydrophobic contorted lipid bilayer segregating two continuous, but nonintersecting water channels are optically isotropic, transparent and stiff viscous gel [1,2]. The water channels have a diameter of about 5 nm in the fully swollen state and the bilayer thickness is roughly 3.5 nm [3]. These cubic phases are stable in excess water and can be fragmented by high energy devices (such as homogenizer, sonicator etc.) into colloidal particles called as cubic nanoparticles or cubosomes [4]. Steric stabilizers such as Pluronics are added to prevent aggregation of fragmented cubic phase particles [5,6]. Cubic nanoparticles are extremely versatile and offer many advantages over other lipid carriers such as emulsions, liposomes, solid lipid nanoparticles and micelle. They have well organized
∗ Corresponding author. Tel.: +91 1662 263515; fax: +91 1662 276240. E-mail addresses: [email protected] (P. Verma), [email protected] (M. Ahuja). http://dx.doi.org/10.1016/j.ijbiomac.2014.10.065 0141-8130/© 2014 Elsevier B.V. All rights reserved.
isotropic structure and large internal area to accommodate significant amount of hydrophilic and/or lipophilic chemical entities. Moreover, the lipid used for fabrication of this delivery system is comparatively inexpensive, biocompatible, biodegradable and non-toxic. Hence, it makes an affordable and attractive delivery vehicle, raising the clinical utility of this strategy [7,8]. Monoolein based cubic nanoparticles are also one of the most widely studied drug delivery applications during the last few years [9–12]. Although, previous studies have revealed exhibition of plausible mucoadhesive properties by monoolein cubic phases, it has also been reported that cubic nanoparticles have very weak and pH dependent interaction with mucin [13,14]. Mucin, a component mucus layer is highly glycosylated to its proline, threonine, and/or serine residues by O-linked N-acetyl galactosamine and N-linked sulfate-bearing glycans. Mucin glycoproteins contain high sialic acid and sulfate content which develop the negative surface charge [15]. The cationic polymers could offer acceptable mucoadhesion via electrostatic interaction. Chitosan, a cationic natural polysaccharide is reported to have excellent mucoadhesion properties both in vitro as well as in vivo [16–21]. Therefore, with the aim of improving mucoadhesion of cubic nanoparticles, chitosan-tailored cubic nanoparticles were designed. Clotrimazole, an antifungal agent was used as a model drug in the chitosan-tailored cubic nanoparticles.
P. Verma, M. Ahuja / International Journal of Biological Macromolecules 73 (2015) 138–145
139
Table 1 Details of central composite design and responses. Runs
Pluronic F127 fraction (% w/w) (X1 )
Chitosan (% w/v) (X2 )
Particle size (nm) (Y1 )
Mucin binding (%) (Y2 )
Zeta potential (mV)
% EE*
1 2 3 4 5 6 7 8 9 10 11 12 13
6(−1) 8(0) 10(+1) 8(0) 8(0) 10(+1) 10(+1) 6(−1) 8(0) 8(0) 8(0) 8(0) 6(−1)
0.5(+1) 0.25(0) 0.25(0) 0.5(+1) 0.25(0) 0.5(+1) 0(−1) 0.25(0) 0.25(0) 0.25(0) 0(−1) 0.25(0) 0(−1)
241.00 86.85 42.51 230.12 101.50 221.40 10.49 88.80 67.65 66.45 15.46 95.05 16.00
3.44 64.13 29.65 40.68 54.17 28.27 15.86 24.13 53.1 60.18 18.18 50.82 11.72
15.8 9.32 10.7 44.2 19.7 23.2 −9.39 26.3 10.1 20.6 −10.15 8.68 −11.6
98.59 ± 1.52 97.89 ± 0.09 99.67 ± 1.11 98.01 ± 1.15 97.86 ± 1.01 99.12 ± 0.08 99.12 ± 1.10 98.62 ± 1.67 97.64 ± 1.26 98.34 ± 1.11 99.11 ± 1.33 98.45 ± 1.11 99.45 ± 1.71
*
Values are mean ± SD (n = 3).
In the present study, chitosan-tailored cubic nanoparticles were formulated employing thin film hydration technique. The formulation variables were optimized using Design of Experiment employing two factors, three levels central composite experimental design. The two independent variables viz fraction of Pluronic F127 to total of monoolein and Pluronic F127 (% w/w) and concentration of chitosan in formulation (% w/v) were studied for their influence on dependent variables-particle size and % mucin binding. Presence of cubic phase was characterized by polarized light microscopy, small-angle X-ray scattering and transmission electron microscopy. Further, comparative antifungal studies were carried out for optimized formulation and conventional clotrimazole suspension.
technique, employed for systematic screening of effect of independent variables on dependent variables. Formulation variable selected as independent variable for optimization were: fraction of Pluronic F127 to total of monoolein and Pluronic F127 (% w/w), and concentration of chitosan in formulation (% w/v). Whereas the response variable were: particle size and % mucin binding. A total of 13 experiments, including 4 factorial points (levels −1 and +1), 4 axial points (levels ±˛), and 5 replicates at the central point (0,0) were generated for estimation of pure error and performed in random order. Details of design are shown in Table 1. The polynomial model fitting quality was evaluated similar to the determination coefficient (R2 ). The experimental design and statistical analysis of data were performed using Design Expert® software (Version 7.0.0, Stat-ease Inc., Minneapolis, MN).
2. Experimental 2.4. HPLC analysis 2.1. Materials Monoolein (RYLO MG 19, monoglycerides content >95%) was a generous gift from Danisco Cultor (Grinsted, Denmark). Clotrimazole and Pluronic F127 (PEO98 –poly PPO67 –PEO98 ) was obtained from Ranbaxy Research Laboratory (Gurgaon, India). Chitosan was provided by Sea Foods (Cochin, India). Sephadex® G-50 was purchased from Sigma-Aldrich (St. Louis, USA). All other chemicals used were of analytical grade. 2.2. Preparation of cubic nanoparticles Briefly, a solution containing varying amounts of monoolein and Pluronic F127 (as per the design protocol) and clotrimazole (0.2%, w/w) in chloroform was prepared in a round bottom flask. The solution so obtained was subjected to evaporation in a Rotary Vacuum Evaporator (Steroglass, Italy) under reduced pressure at 50 ◦ C to obtain a thin film on inner surface of round bottom flask. The thin film so obtained was hydrated with purified water or chitosan solution (0.25 or 0.5, % w/v) to obtain dispersion. Chitosan solution used for hydration of film was prepared by dissolving the required quantity of chitosan in acetic acid (1%, v/v). Thereafter, coarse dispersion was sonicated for 1 h at temperature 60 ◦ C in a bath ultrasonicator (Powersonic405, Hwashin Technology Co., Korea) to obtain cubic nanoparticles. 2.3. Experimental design In the present work, the preparation of clotimazole-loaded cubic nanoparticles was optimized using response surface methodology (RSM) employing 2-factor, 3-level Central Composite Design (with ˛ = 1). Central Composite Design is a multivariate statistic
The assay of clotrimazole in samples, was carried out by injecting 20 l of the solution, spiked with metronidazole (as internal standard), into a chromatographic system equipped with 600 pump controller (Waters, USA), 2487 dual wavelength absorbance UV detector (Waters, USA) and 7725i manual injector (Rheodyne, USA). The chromatographic separation of clotrimazole was achieved employing mobile phase composition of methanol:water (80:20, v/v) at a flow rate of 0.8 ml/min, in an isocratic run through SpherisorbC18 column. The eluent was analysed for clotrimazole at 210 nm. The peak height ratios of clotrimazole to metronidazole exhibited linear correlation with the concentration of clotrimazole with the equation of line, Y = 0.007X + 0.293 (R2 = 0.993). The retention time of metronidazole and clotrimazole was 3.2 min and 10.6 min, respectively. The method was found to be precise as the results of intermediate precision and repeatability studies showed relative standard deviation
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Optimization, characterization and evaluation of chitosan-tailored cubic nanoparticles of clotrimazole Purnima Verma, Munish Ahuja ∗ Drug Delivery Research Laboratory, Department of Pharmaceutical Sciences, Guru Jambheshwar University of Science and Technology, Delhi-Bye pass Road, Hisar 125 001, Haryana, India
a r t i c l e
i n f o
Article history: Received 27 August 2014 Received in revised form 18 October 2014 Accepted 30 October 2014 Available online 24 November 2014 Keywords: Cubic nanoparticles Chitosan Central composite design
a b s t r a c t The present study deals with improvement of the mucoadhesive properties of monoolein based cubic nanoparticles by incorporating chitosan. Chitosan-tailored cubic nanoparticles were prepared by thin film hydration followed by ultrasonication employing clotrimazole as model drug. The effect of Pluronic F127 fraction and concentration of chitosan on particle size and % mucin binding of the formulations was studied using 2-factor, 3-level, central composite experimental design. The concentration of chitosan was found to influence particle size and % mucin binding of cubic nanoparticles while Pluronic F127 fraction influenced only the % mucin binding. Studies indicated 8.33(%w/w) fraction of Pluronic F127 and 0.17 (%w/v) concentration of chitosan as optimum concentration. Finally, the optimized batch was characterized by polarized light microscopy, small-angle X-ray scattering (SAXS) and transmission electron microscopy. The results unveiled incorporation of chitosan did not disrupt the inner cubic structure of nanoparticles. Peak indexing of SAXS data revealed the coexistence of P-type and D-type cubic phases in nanoparticles. Further, comparative evaluation studies showed significantly higher anti-fungal activity of clotrimazole-loaded chitosan-tailored cubic nanoparticles than conventional suspension of clotrimazole against Candida albicans. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Monoolein is a polar unsaturated long chain monoglyceride. It can self-associate in water to form thermodynamically stable, well defined cubic phases. The cubic phases which comprises of hydrophobic contorted lipid bilayer segregating two continuous, but nonintersecting water channels are optically isotropic, transparent and stiff viscous gel [1,2]. The water channels have a diameter of about 5 nm in the fully swollen state and the bilayer thickness is roughly 3.5 nm [3]. These cubic phases are stable in excess water and can be fragmented by high energy devices (such as homogenizer, sonicator etc.) into colloidal particles called as cubic nanoparticles or cubosomes [4]. Steric stabilizers such as Pluronics are added to prevent aggregation of fragmented cubic phase particles [5,6]. Cubic nanoparticles are extremely versatile and offer many advantages over other lipid carriers such as emulsions, liposomes, solid lipid nanoparticles and micelle. They have well organized
∗ Corresponding author. Tel.: +91 1662 263515; fax: +91 1662 276240. E-mail addresses: [email protected] (P. Verma), [email protected] (M. Ahuja). http://dx.doi.org/10.1016/j.ijbiomac.2014.10.065 0141-8130/© 2014 Elsevier B.V. All rights reserved.
isotropic structure and large internal area to accommodate significant amount of hydrophilic and/or lipophilic chemical entities. Moreover, the lipid used for fabrication of this delivery system is comparatively inexpensive, biocompatible, biodegradable and non-toxic. Hence, it makes an affordable and attractive delivery vehicle, raising the clinical utility of this strategy [7,8]. Monoolein based cubic nanoparticles are also one of the most widely studied drug delivery applications during the last few years [9–12]. Although, previous studies have revealed exhibition of plausible mucoadhesive properties by monoolein cubic phases, it has also been reported that cubic nanoparticles have very weak and pH dependent interaction with mucin [13,14]. Mucin, a component mucus layer is highly glycosylated to its proline, threonine, and/or serine residues by O-linked N-acetyl galactosamine and N-linked sulfate-bearing glycans. Mucin glycoproteins contain high sialic acid and sulfate content which develop the negative surface charge [15]. The cationic polymers could offer acceptable mucoadhesion via electrostatic interaction. Chitosan, a cationic natural polysaccharide is reported to have excellent mucoadhesion properties both in vitro as well as in vivo [16–21]. Therefore, with the aim of improving mucoadhesion of cubic nanoparticles, chitosan-tailored cubic nanoparticles were designed. Clotrimazole, an antifungal agent was used as a model drug in the chitosan-tailored cubic nanoparticles.
P. Verma, M. Ahuja / International Journal of Biological Macromolecules 73 (2015) 138–145
139
Table 1 Details of central composite design and responses. Runs
Pluronic F127 fraction (% w/w) (X1 )
Chitosan (% w/v) (X2 )
Particle size (nm) (Y1 )
Mucin binding (%) (Y2 )
Zeta potential (mV)
% EE*
1 2 3 4 5 6 7 8 9 10 11 12 13
6(−1) 8(0) 10(+1) 8(0) 8(0) 10(+1) 10(+1) 6(−1) 8(0) 8(0) 8(0) 8(0) 6(−1)
0.5(+1) 0.25(0) 0.25(0) 0.5(+1) 0.25(0) 0.5(+1) 0(−1) 0.25(0) 0.25(0) 0.25(0) 0(−1) 0.25(0) 0(−1)
241.00 86.85 42.51 230.12 101.50 221.40 10.49 88.80 67.65 66.45 15.46 95.05 16.00
3.44 64.13 29.65 40.68 54.17 28.27 15.86 24.13 53.1 60.18 18.18 50.82 11.72
15.8 9.32 10.7 44.2 19.7 23.2 −9.39 26.3 10.1 20.6 −10.15 8.68 −11.6
98.59 ± 1.52 97.89 ± 0.09 99.67 ± 1.11 98.01 ± 1.15 97.86 ± 1.01 99.12 ± 0.08 99.12 ± 1.10 98.62 ± 1.67 97.64 ± 1.26 98.34 ± 1.11 99.11 ± 1.33 98.45 ± 1.11 99.45 ± 1.71
*
Values are mean ± SD (n = 3).
In the present study, chitosan-tailored cubic nanoparticles were formulated employing thin film hydration technique. The formulation variables were optimized using Design of Experiment employing two factors, three levels central composite experimental design. The two independent variables viz fraction of Pluronic F127 to total of monoolein and Pluronic F127 (% w/w) and concentration of chitosan in formulation (% w/v) were studied for their influence on dependent variables-particle size and % mucin binding. Presence of cubic phase was characterized by polarized light microscopy, small-angle X-ray scattering and transmission electron microscopy. Further, comparative antifungal studies were carried out for optimized formulation and conventional clotrimazole suspension.
technique, employed for systematic screening of effect of independent variables on dependent variables. Formulation variable selected as independent variable for optimization were: fraction of Pluronic F127 to total of monoolein and Pluronic F127 (% w/w), and concentration of chitosan in formulation (% w/v). Whereas the response variable were: particle size and % mucin binding. A total of 13 experiments, including 4 factorial points (levels −1 and +1), 4 axial points (levels ±˛), and 5 replicates at the central point (0,0) were generated for estimation of pure error and performed in random order. Details of design are shown in Table 1. The polynomial model fitting quality was evaluated similar to the determination coefficient (R2 ). The experimental design and statistical analysis of data were performed using Design Expert® software (Version 7.0.0, Stat-ease Inc., Minneapolis, MN).
2. Experimental 2.4. HPLC analysis 2.1. Materials Monoolein (RYLO MG 19, monoglycerides content >95%) was a generous gift from Danisco Cultor (Grinsted, Denmark). Clotrimazole and Pluronic F127 (PEO98 –poly PPO67 –PEO98 ) was obtained from Ranbaxy Research Laboratory (Gurgaon, India). Chitosan was provided by Sea Foods (Cochin, India). Sephadex® G-50 was purchased from Sigma-Aldrich (St. Louis, USA). All other chemicals used were of analytical grade. 2.2. Preparation of cubic nanoparticles Briefly, a solution containing varying amounts of monoolein and Pluronic F127 (as per the design protocol) and clotrimazole (0.2%, w/w) in chloroform was prepared in a round bottom flask. The solution so obtained was subjected to evaporation in a Rotary Vacuum Evaporator (Steroglass, Italy) under reduced pressure at 50 ◦ C to obtain a thin film on inner surface of round bottom flask. The thin film so obtained was hydrated with purified water or chitosan solution (0.25 or 0.5, % w/v) to obtain dispersion. Chitosan solution used for hydration of film was prepared by dissolving the required quantity of chitosan in acetic acid (1%, v/v). Thereafter, coarse dispersion was sonicated for 1 h at temperature 60 ◦ C in a bath ultrasonicator (Powersonic405, Hwashin Technology Co., Korea) to obtain cubic nanoparticles. 2.3. Experimental design In the present work, the preparation of clotimazole-loaded cubic nanoparticles was optimized using response surface methodology (RSM) employing 2-factor, 3-level Central Composite Design (with ˛ = 1). Central Composite Design is a multivariate statistic
The assay of clotrimazole in samples, was carried out by injecting 20 l of the solution, spiked with metronidazole (as internal standard), into a chromatographic system equipped with 600 pump controller (Waters, USA), 2487 dual wavelength absorbance UV detector (Waters, USA) and 7725i manual injector (Rheodyne, USA). The chromatographic separation of clotrimazole was achieved employing mobile phase composition of methanol:water (80:20, v/v) at a flow rate of 0.8 ml/min, in an isocratic run through SpherisorbC18 column. The eluent was analysed for clotrimazole at 210 nm. The peak height ratios of clotrimazole to metronidazole exhibited linear correlation with the concentration of clotrimazole with the equation of line, Y = 0.007X + 0.293 (R2 = 0.993). The retention time of metronidazole and clotrimazole was 3.2 min and 10.6 min, respectively. The method was found to be precise as the results of intermediate precision and repeatability studies showed relative standard deviation
