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Pharmacokinetic evaluation of testosterone-loaded nanocapsules in rats
International Journal of Biological Macromolecules 72 (2015) 28–30
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Short communication
Pharmacokinetic evaluation of testosterone-loaded nanocapsules in rats Sougata Jana a,∗ , Anusuya Gangopadhaya b , Benoy Brata Bhowmik b , Amit Kumar Nayak c , Arup Mukherjee b a
Department of Pharmaceutics, Gupta College of Technological Sciences, Asansol 713301, WB, India Division of Pharmaceutical Technology, Department of Chemical Technology, University of Calcutta, 92, A.P.C. Road, Kolkata 700009, WB, India c Department of Pharmaceutics, Seemanta Institute of Pharmaceutical Sciences, Mayurbhanj 757086, Odisha, India b
a r t i c l e
i n f o
Article history: Received 26 June 2014 Received in revised form 26 July 2014 Accepted 3 August 2014 Available online 8 August 2014 Keywords: Alginate Nanocapsule Testosterone
a b s t r a c t In the present work, testosterone-loaded alginate nanocapsules were prepared by in situ nanoemulsification-polymer cross-linking method. Drug loading, particle size (diameter), polydispersity index and zeta potential of these alginate nanocapsules were measured 30.22 ± 1.90%, 34.53 ± 1.72 nm, 0.22 ± 0.04 and −5 mV, respectively. The pharmacokinetic evaluation of these nanocapsules was also performed in female Sprague Dawley rats. Cmax , Tmax and AUC0–24 vales were estimated 38.63 ng/ml, 2 h and 317.93 ng ml−1 h−1 , respectively. The pharmacokinetic result of testosterone-loaded alginate nanocapsules indicates better bioavailability in comparison with pure testosterone and commercial testosterone injection. © 2014 Published by Elsevier B.V.
1. Introduction Recent years, a considerable research interest has been observed in the field of nanoparticle-based drug delivery systems [1–3]. Among various types of nanoparticles, polymeric nano-systems have received lots of attention due to their stability and ease of surface modification specificity [4–8]. Polymeric nanocapsules are one of the varieties of nano-systems in which drug particles are encapsulated by biocompatible and biodegradable polymers [6,8]. Nanocapsules within a certain size range are known to be easily circulating, passing even through small capillaries without clogging [9]. Testosterone is a steroidal male hormone that regulates cellular calcium transport [10]. It is used in the treatment of osteoporosis and coronary artery diseases [11]. Testosterone is also useful in suppressing diverse immune reactions and gerontology [12,13]. Health implications of testosterone and also its large-scale use by athletes have propelled investigations on different modified drug delivery systems for testosterone [14]. Testosterone has a relatively short plasma half-life of about 10–20 min [15,16]. It undergoes extensive first-pass metabolism in liver [17]. Because of these limitations, a sustained testosterone release drug delivery would be
∗ Corresponding author. Tel.: +91 9434896683. E-mail address: [email protected] (S. Jana). http://dx.doi.org/10.1016/j.ijbiomac.2014.08.001 0141-8130/© 2014 Published by Elsevier B.V.
beneficial. In a previous investigation, we have formulated alginate nanocapsules containing testosterone and evaluated its in vitro sustained release [9]. However, the pharmacokinetic performances of these newly formulated testosterone-loaded nanocapsules are not explored till now. Therefore in this investigation, we made an attempt to evaluate pharmacokinetic parameters of testosteroneloaded alginate nanocapsules in rats. 2. Materials and method 2.1. Materials Testosterone (C19 H28 O2 ) was obtained as a gift sample from Organon India Ltd., India. Sodium alginate (250 cps viscosity, Sigma Chemicals, USA) and calcium chloride (Spectrochem Pvt. Ltd., India) were used. All organic solvents and water used were of high performance liquid chromatography (HPLC) grade (Merck/Spectrochem Pvt. Ltd., India). All other reagents were of analytical grade and procured from commercial sources. 2.2. Preparation of testosterone-loaded alginate nanocapsules Testosterone-loaded alginate nanocapsules were prepared through in situ nanoemulsification-polymer cross-linking method. Briefly, testosterone (5 mg) was dissolved in 10 ml hexane and emulsified into 30 ml 0.1% (w/v) aqueous solution of sodium
S. Jana et al. / International Journal of Biological Macromolecules 72 (2015) 28–30
29
alginate using Tween 80 (0.09 mg) as emulsifier under stirring (500 rpm) followed by sonication (20 KHz). Stabilizer, glycerol (∼30 ml) was then added drop-wise (0.1 ml/min) into nanoemulsion. Calcium chloride solution (2 M CaCl2 ; 1.5 ml) was added into the reaction mixture to effect cross-linking to produce nanocapsules. The reaction mixture was cured for 24 h at room temperature (25 ◦ C). Nanocapsules were then separated by ultracentrifugation (Sorvall Ultra 80, Sorvall, USA) at 30,000 rpm, 0 ◦ C for 30 min. Nanocapsules thus, obtained were washed with 15 ml of water, recentrifuged and harvested in microcentrifuge tubes. There were then preserved in vaccum desiccators at 4 ◦ C for further evaluations. The drug to polymer ratio was maintained 1:6 (w/w). 2.3. Evaluation of testosterone Reverse-phase HPLC method was used to determine the estimation of testosterone. The HPLC set-up used consisted of LC-6A, Shimadzu System (Japan) with a UV–vis detector and ODS column (Zorbax-ODS, Aligent, USA). The mobile-phase used was methanol–water (80:20) at a flow-rate of 0.5 ml/min. Elluents were monitored at a fixed max of 239 nm checked previously in a UV spectrophotometer (U-2000, Hitachi, Japan). A 20 l sample volume was injected each time through a Rheodyne injector and the HPLC peak areas were recovered from the chromatogram. HPLC elution time for testosterone was measured 12 min. A standard curve was first prepared using known concentrations of standard testosterone against the HPLC peak area and was used through out the analysis. Standard curve equation was y = 5.32 × 106 x + 686.03, R2 = 0.9998 (where, y = HPLC peak area, x = concentration in mg/ml, R = Correlation of co-efficient). 2.4. Determination of drug loading Accurately weighed, 20 mg testosterone-loaded nanocapsules were digested in 40 ml of 1% (w/v) sodium citrate solution and final volume was made up to 50 ml with deionized water. The mixture was sonicated at 20 KHz for 30 min and the solution was filtered using centricon tubes (molecular mass cut-off 50,000) in a cold centrifuge (Remi C30, India) at 0 ◦ C, 10,000 rpm. 20 l of filtrate was injected into HPLC and the drug loading was calculated as: Drug loading(%) =
testosterone released on digestion (mg) × 100 testosterone taken (mg)
2.5. Particle size, polydispersity index and zeta potential determination Particle size, polydispersity index and zeta potential were determined using large bore capillary cells in the Zetasizer Nano-ZS (Malvern Instruments). 1 ml of testosterone-loaded nanocapsules containing suspension from the preparation medium was samples out and diluted to 8 ml with 0.9% (w/v) sodium chloride solution prepared in HPLC water for optimal signal intensity [18]. The pH of the water used was 6.0 ± 0.1. 2.6. Pharmacokinetic evaluation The experiment was approved by Institutional Animal Ethical Committee, under registration number 506/01/a/CPCSEA. Female Sprague Dawley rats (200–225 g) were used for this study. The animals were given ad libitum access to water and bengal gram as food. Animals were divided randomly into 4 groups of 7 rats each and treated as follows: Group A: control, Group B: testosterone-loaded alginate nanocapsules, Group C: pure testosterone and Group D: commercial testosterone injection. In Groups A–C, water for injection IP was used as vehicle. Animals of different groups were treated
Fig. 1. Testosterone plasma concentration vs. time curve.
by subcutaneous injection at a dose equivalent to 50 mg testosterone/kg body weight. Blood samples were collected from cardiac puncture under anesthesia using ether. Then, the blood serums of different groups were separated by centrifugation for 5 min at 3000 rpm and collected blood serums were stored at −20 ◦ C until analyze by Automated Chemiluminescence’s System (ACS180, Bayer Health Care). 3. Results and discussion In the current work, testosterone-loaded alginate nanocapsules were prepared through in situ nanoemulsification-polymer crosslinking using hexane as a solvent. The loading of testosterone in alginate nanocapsules was found 30.22 ± 1.90%. The particle diameter (z-average diameter) of prepared nanocapsules was measured 34.53 ± 1.72 nm. The polydispersity index of these nanocapsules was recorded 0.22 ± 0.04. It was established that polydispersity index near 0.25 is generally considered ideal and suitable for long circulation in injectable preparations [9,19]. It was considered that extremely positive or negative zeta potential values cause larger repulsive forces, while electrostatic repulsion between particles with the same electric charge prevents aggregation of the particles [20]. The zeta potential of nanocapsules was measured −5 mV. Thus, the prepared testosterone-loaded alginate nanocapsules were of nano-sized, suitable for long circulating nanocapsules in injectable preparations and free from agglomeration. Pharmacokinetic evaluation of testosterone-loaded alginate nanocapsules was performed in Female Sprague Dawley rats. The testosterone plasma concentration vs. time curve for different estimated groups was presented in Fig. 1. Various important pharmacokinetic parameters Cmax , Tmax and AUC0–24 were also estimated and presented in Table 1. Though Tmax for Group B, C and D was same (i.e., 2 h), Cmax and AUC0–24 of prepared testosteroneloaded alginate nanocapsules were higher (38.63 ng/ml and 317.93 ng ml−1 h−1 , respectively) than other investigated groups in the study, indicating better bioavailability by the testosteroneloaded alginate nanocapsules in comparison with pure testosterone Table 1 Pharmacokinetic parameters of testosterone-loaded alginate nanocapsules in rats. Groups treated with
Cmax (ng/ml)
Tmax (h)
AUC0–24 (ng ml−1 h−1 )
Testosterone-loaded alginate nanocapsules Pure testosterone Commercial testosterone injection
38.63
2.1
317.93
21.72 18.90
1.98 2.01
201.08 175.70
30
S. Jana et al. / International Journal of Biological Macromolecules 72 (2015) 28–30
and commercial testosterone injection in the pharmacokinetic study. 4. Conclusion Testosterone-loaded alginate nanocapsules with testosterone loading of 30.22 ± 1.90% were prepared by in situ nanoemulsification-polymer cross-linking method. Various physicochemical parameters like particle size (diameter), polydispersity index and zeta potential were measured 34.53 ± 1.72 nm, 0.22 ± 0.04 and −5 mV, respectively, which indicate nano-sized diameter of nanocapsules, suitability for long circulating in injectable preparations and free from agglomeration. The pharmacokinetic performance of these testosterone-loaded alginate nanocapsules were also evaluated in Female Sprague Dawley rats, indicating better bioavailability in comparison with pure testosterone and commercial testosterone injection. References [1] J. Malakar, A. Ghosh, A. Basu, A.K. Nayak, Int. Res. J. Pharm. 3 (2012) 36–40. [2] A.K. Nayak, A.K. Dhara, Arch. Appl. Sci. Res. 2 (2010) 284–293. [3] D. Pal, A.K. Nayak, Int. J. Pharm. Sci. Rev. Res. 1 (2010) 1–7.
[4] X.D. Yang, Q.Q. Zhang, Y.S. Wang, H. Chen, H.Z. Zhang, F.P. Gao, L.R. Lin, Colloids Surf. B: Biointerfaces 61 (2008) 125–131. [5] M.R. Sobaktakin, R.M. Tabatabaie, M.A. Ramazanov, Int. J. Biol. Macromol. 48 (2011) 403–407. [6] H. Pawar, D. Douroumis, J.S. Boateng, Colloids Surf. B: Biointerfaces 90 (2012) 102–108. [7] S. Jana, S. Manna, A.K. Nayak, K.K. Sen, S.K. Basu, Colloids Surf. B: Biointerfaces 114 (2014) 36–44. [8] S. Jana, N. Maji, A.K. Nayak, K.K. Sen, S.K. Basu, Carbohydr. Polym. 98 (2013) 870–876. [9] B. Bhowmik, B. Sa, A. Mukherjee, Acta Pharm. 56 (2006) 417–429. [10] P.J. Synder, in: G.J. Hardman, E.L. Limbards (Eds.), Goodman and Gilman’s The Pharmacological Basis of Therapeutics, 10th ed., Mc Graw-Hill Companies Inc., New York, 2001, pp. 1635–1648. [11] G.B. Phillips, B.H. Pinkernell, T.Y. Jing, Arterioscler. Thromb. 14 (1994) 701–706. [12] A. Bouman, M.J. Heineman, M.M. Faas, Hum. Reprod. Update 11 (2005) 411–423. [13] I.M. Chapman, Best Pract. Res.: Clin. Endocrinol. Metab. 18 (2004) 437–452. [14] S. Kim, W. Snipes, G.D. Hodgen, F. Anderson, Contraception 52 (1995) 313–316. [15] British Pharmacopoeia, vol. I, British Pharmacopoeia Commission, London, 1993, pp. 654. [16] Martindale, The Extra Pharmacopoeia, 29th ed., The Pharmaceutical Press, London, 1989, pp. 1414. [17] S. Jay, W. Fountain, Z. Cui, R.J. Mumper, J. Pharm. Sci. 91 (2002) 2016–2025. [18] J. Vandervoort, A. Ludwig, Eur. J. Pharm. Biopharm. 57 (2004) 251–261. ¨` [19] R.H. Muller, S. Schmidt, I. Buttle, A. Akkar, J. Schmitt, S. Brömer, Int. J. Pharm. 269 (2004) 293–302. [20] N. Passerini, D.Q.M. Craig, J. Pharm. Pharmacol. 54 (2002) 913–919.
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Short communication
Pharmacokinetic evaluation of testosterone-loaded nanocapsules in rats Sougata Jana a,∗ , Anusuya Gangopadhaya b , Benoy Brata Bhowmik b , Amit Kumar Nayak c , Arup Mukherjee b a
Department of Pharmaceutics, Gupta College of Technological Sciences, Asansol 713301, WB, India Division of Pharmaceutical Technology, Department of Chemical Technology, University of Calcutta, 92, A.P.C. Road, Kolkata 700009, WB, India c Department of Pharmaceutics, Seemanta Institute of Pharmaceutical Sciences, Mayurbhanj 757086, Odisha, India b
a r t i c l e
i n f o
Article history: Received 26 June 2014 Received in revised form 26 July 2014 Accepted 3 August 2014 Available online 8 August 2014 Keywords: Alginate Nanocapsule Testosterone
a b s t r a c t In the present work, testosterone-loaded alginate nanocapsules were prepared by in situ nanoemulsification-polymer cross-linking method. Drug loading, particle size (diameter), polydispersity index and zeta potential of these alginate nanocapsules were measured 30.22 ± 1.90%, 34.53 ± 1.72 nm, 0.22 ± 0.04 and −5 mV, respectively. The pharmacokinetic evaluation of these nanocapsules was also performed in female Sprague Dawley rats. Cmax , Tmax and AUC0–24 vales were estimated 38.63 ng/ml, 2 h and 317.93 ng ml−1 h−1 , respectively. The pharmacokinetic result of testosterone-loaded alginate nanocapsules indicates better bioavailability in comparison with pure testosterone and commercial testosterone injection. © 2014 Published by Elsevier B.V.
1. Introduction Recent years, a considerable research interest has been observed in the field of nanoparticle-based drug delivery systems [1–3]. Among various types of nanoparticles, polymeric nano-systems have received lots of attention due to their stability and ease of surface modification specificity [4–8]. Polymeric nanocapsules are one of the varieties of nano-systems in which drug particles are encapsulated by biocompatible and biodegradable polymers [6,8]. Nanocapsules within a certain size range are known to be easily circulating, passing even through small capillaries without clogging [9]. Testosterone is a steroidal male hormone that regulates cellular calcium transport [10]. It is used in the treatment of osteoporosis and coronary artery diseases [11]. Testosterone is also useful in suppressing diverse immune reactions and gerontology [12,13]. Health implications of testosterone and also its large-scale use by athletes have propelled investigations on different modified drug delivery systems for testosterone [14]. Testosterone has a relatively short plasma half-life of about 10–20 min [15,16]. It undergoes extensive first-pass metabolism in liver [17]. Because of these limitations, a sustained testosterone release drug delivery would be
∗ Corresponding author. Tel.: +91 9434896683. E-mail address: [email protected] (S. Jana). http://dx.doi.org/10.1016/j.ijbiomac.2014.08.001 0141-8130/© 2014 Published by Elsevier B.V.
beneficial. In a previous investigation, we have formulated alginate nanocapsules containing testosterone and evaluated its in vitro sustained release [9]. However, the pharmacokinetic performances of these newly formulated testosterone-loaded nanocapsules are not explored till now. Therefore in this investigation, we made an attempt to evaluate pharmacokinetic parameters of testosteroneloaded alginate nanocapsules in rats. 2. Materials and method 2.1. Materials Testosterone (C19 H28 O2 ) was obtained as a gift sample from Organon India Ltd., India. Sodium alginate (250 cps viscosity, Sigma Chemicals, USA) and calcium chloride (Spectrochem Pvt. Ltd., India) were used. All organic solvents and water used were of high performance liquid chromatography (HPLC) grade (Merck/Spectrochem Pvt. Ltd., India). All other reagents were of analytical grade and procured from commercial sources. 2.2. Preparation of testosterone-loaded alginate nanocapsules Testosterone-loaded alginate nanocapsules were prepared through in situ nanoemulsification-polymer cross-linking method. Briefly, testosterone (5 mg) was dissolved in 10 ml hexane and emulsified into 30 ml 0.1% (w/v) aqueous solution of sodium
S. Jana et al. / International Journal of Biological Macromolecules 72 (2015) 28–30
29
alginate using Tween 80 (0.09 mg) as emulsifier under stirring (500 rpm) followed by sonication (20 KHz). Stabilizer, glycerol (∼30 ml) was then added drop-wise (0.1 ml/min) into nanoemulsion. Calcium chloride solution (2 M CaCl2 ; 1.5 ml) was added into the reaction mixture to effect cross-linking to produce nanocapsules. The reaction mixture was cured for 24 h at room temperature (25 ◦ C). Nanocapsules were then separated by ultracentrifugation (Sorvall Ultra 80, Sorvall, USA) at 30,000 rpm, 0 ◦ C for 30 min. Nanocapsules thus, obtained were washed with 15 ml of water, recentrifuged and harvested in microcentrifuge tubes. There were then preserved in vaccum desiccators at 4 ◦ C for further evaluations. The drug to polymer ratio was maintained 1:6 (w/w). 2.3. Evaluation of testosterone Reverse-phase HPLC method was used to determine the estimation of testosterone. The HPLC set-up used consisted of LC-6A, Shimadzu System (Japan) with a UV–vis detector and ODS column (Zorbax-ODS, Aligent, USA). The mobile-phase used was methanol–water (80:20) at a flow-rate of 0.5 ml/min. Elluents were monitored at a fixed max of 239 nm checked previously in a UV spectrophotometer (U-2000, Hitachi, Japan). A 20 l sample volume was injected each time through a Rheodyne injector and the HPLC peak areas were recovered from the chromatogram. HPLC elution time for testosterone was measured 12 min. A standard curve was first prepared using known concentrations of standard testosterone against the HPLC peak area and was used through out the analysis. Standard curve equation was y = 5.32 × 106 x + 686.03, R2 = 0.9998 (where, y = HPLC peak area, x = concentration in mg/ml, R = Correlation of co-efficient). 2.4. Determination of drug loading Accurately weighed, 20 mg testosterone-loaded nanocapsules were digested in 40 ml of 1% (w/v) sodium citrate solution and final volume was made up to 50 ml with deionized water. The mixture was sonicated at 20 KHz for 30 min and the solution was filtered using centricon tubes (molecular mass cut-off 50,000) in a cold centrifuge (Remi C30, India) at 0 ◦ C, 10,000 rpm. 20 l of filtrate was injected into HPLC and the drug loading was calculated as: Drug loading(%) =
testosterone released on digestion (mg) × 100 testosterone taken (mg)
2.5. Particle size, polydispersity index and zeta potential determination Particle size, polydispersity index and zeta potential were determined using large bore capillary cells in the Zetasizer Nano-ZS (Malvern Instruments). 1 ml of testosterone-loaded nanocapsules containing suspension from the preparation medium was samples out and diluted to 8 ml with 0.9% (w/v) sodium chloride solution prepared in HPLC water for optimal signal intensity [18]. The pH of the water used was 6.0 ± 0.1. 2.6. Pharmacokinetic evaluation The experiment was approved by Institutional Animal Ethical Committee, under registration number 506/01/a/CPCSEA. Female Sprague Dawley rats (200–225 g) were used for this study. The animals were given ad libitum access to water and bengal gram as food. Animals were divided randomly into 4 groups of 7 rats each and treated as follows: Group A: control, Group B: testosterone-loaded alginate nanocapsules, Group C: pure testosterone and Group D: commercial testosterone injection. In Groups A–C, water for injection IP was used as vehicle. Animals of different groups were treated
Fig. 1. Testosterone plasma concentration vs. time curve.
by subcutaneous injection at a dose equivalent to 50 mg testosterone/kg body weight. Blood samples were collected from cardiac puncture under anesthesia using ether. Then, the blood serums of different groups were separated by centrifugation for 5 min at 3000 rpm and collected blood serums were stored at −20 ◦ C until analyze by Automated Chemiluminescence’s System (ACS180, Bayer Health Care). 3. Results and discussion In the current work, testosterone-loaded alginate nanocapsules were prepared through in situ nanoemulsification-polymer crosslinking using hexane as a solvent. The loading of testosterone in alginate nanocapsules was found 30.22 ± 1.90%. The particle diameter (z-average diameter) of prepared nanocapsules was measured 34.53 ± 1.72 nm. The polydispersity index of these nanocapsules was recorded 0.22 ± 0.04. It was established that polydispersity index near 0.25 is generally considered ideal and suitable for long circulation in injectable preparations [9,19]. It was considered that extremely positive or negative zeta potential values cause larger repulsive forces, while electrostatic repulsion between particles with the same electric charge prevents aggregation of the particles [20]. The zeta potential of nanocapsules was measured −5 mV. Thus, the prepared testosterone-loaded alginate nanocapsules were of nano-sized, suitable for long circulating nanocapsules in injectable preparations and free from agglomeration. Pharmacokinetic evaluation of testosterone-loaded alginate nanocapsules was performed in Female Sprague Dawley rats. The testosterone plasma concentration vs. time curve for different estimated groups was presented in Fig. 1. Various important pharmacokinetic parameters Cmax , Tmax and AUC0–24 were also estimated and presented in Table 1. Though Tmax for Group B, C and D was same (i.e., 2 h), Cmax and AUC0–24 of prepared testosteroneloaded alginate nanocapsules were higher (38.63 ng/ml and 317.93 ng ml−1 h−1 , respectively) than other investigated groups in the study, indicating better bioavailability by the testosteroneloaded alginate nanocapsules in comparison with pure testosterone Table 1 Pharmacokinetic parameters of testosterone-loaded alginate nanocapsules in rats. Groups treated with
Cmax (ng/ml)
Tmax (h)
AUC0–24 (ng ml−1 h−1 )
Testosterone-loaded alginate nanocapsules Pure testosterone Commercial testosterone injection
38.63
2.1
317.93
21.72 18.90
1.98 2.01
201.08 175.70
30
S. Jana et al. / International Journal of Biological Macromolecules 72 (2015) 28–30
and commercial testosterone injection in the pharmacokinetic study. 4. Conclusion Testosterone-loaded alginate nanocapsules with testosterone loading of 30.22 ± 1.90% were prepared by in situ nanoemulsification-polymer cross-linking method. Various physicochemical parameters like particle size (diameter), polydispersity index and zeta potential were measured 34.53 ± 1.72 nm, 0.22 ± 0.04 and −5 mV, respectively, which indicate nano-sized diameter of nanocapsules, suitability for long circulating in injectable preparations and free from agglomeration. The pharmacokinetic performance of these testosterone-loaded alginate nanocapsules were also evaluated in Female Sprague Dawley rats, indicating better bioavailability in comparison with pure testosterone and commercial testosterone injection. References [1] J. Malakar, A. Ghosh, A. Basu, A.K. Nayak, Int. Res. J. Pharm. 3 (2012) 36–40. [2] A.K. Nayak, A.K. Dhara, Arch. Appl. Sci. Res. 2 (2010) 284–293. [3] D. Pal, A.K. Nayak, Int. J. Pharm. Sci. Rev. Res. 1 (2010) 1–7.
[4] X.D. Yang, Q.Q. Zhang, Y.S. Wang, H. Chen, H.Z. Zhang, F.P. Gao, L.R. Lin, Colloids Surf. B: Biointerfaces 61 (2008) 125–131. [5] M.R. Sobaktakin, R.M. Tabatabaie, M.A. Ramazanov, Int. J. Biol. Macromol. 48 (2011) 403–407. [6] H. Pawar, D. Douroumis, J.S. Boateng, Colloids Surf. B: Biointerfaces 90 (2012) 102–108. [7] S. Jana, S. Manna, A.K. Nayak, K.K. Sen, S.K. Basu, Colloids Surf. B: Biointerfaces 114 (2014) 36–44. [8] S. Jana, N. Maji, A.K. Nayak, K.K. Sen, S.K. Basu, Carbohydr. Polym. 98 (2013) 870–876. [9] B. Bhowmik, B. Sa, A. Mukherjee, Acta Pharm. 56 (2006) 417–429. [10] P.J. Synder, in: G.J. Hardman, E.L. Limbards (Eds.), Goodman and Gilman’s The Pharmacological Basis of Therapeutics, 10th ed., Mc Graw-Hill Companies Inc., New York, 2001, pp. 1635–1648. [11] G.B. Phillips, B.H. Pinkernell, T.Y. Jing, Arterioscler. Thromb. 14 (1994) 701–706. [12] A. Bouman, M.J. Heineman, M.M. Faas, Hum. Reprod. Update 11 (2005) 411–423. [13] I.M. Chapman, Best Pract. Res.: Clin. Endocrinol. Metab. 18 (2004) 437–452. [14] S. Kim, W. Snipes, G.D. Hodgen, F. Anderson, Contraception 52 (1995) 313–316. [15] British Pharmacopoeia, vol. I, British Pharmacopoeia Commission, London, 1993, pp. 654. [16] Martindale, The Extra Pharmacopoeia, 29th ed., The Pharmaceutical Press, London, 1989, pp. 1414. [17] S. Jay, W. Fountain, Z. Cui, R.J. Mumper, J. Pharm. Sci. 91 (2002) 2016–2025. [18] J. Vandervoort, A. Ludwig, Eur. J. Pharm. Biopharm. 57 (2004) 251–261. ¨` [19] R.H. Muller, S. Schmidt, I. Buttle, A. Akkar, J. Schmitt, S. Brömer, Int. J. Pharm. 269 (2004) 293–302. [20] N. Passerini, D.Q.M. Craig, J. Pharm. Pharmacol. 54 (2002) 913–919.