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Formulation and optimization of Embelin nanosuspensions using central composite design for dissolution enhancement
Journal of Drug Delivery Science and Technology 29 (2015) 1e7
Contents lists available at ScienceDirect
Journal of Drug Delivery Science and Technology journal homepage: www.elsevier.com/locate/jddst
Original research
Formulation and optimization of Embelin nanosuspensions using central composite design for dissolution enhancement Komal Parmar a, *, Jayvadan Patel b, Navin Sheth a a b
Department of Pharmaceutical Sciences, Saurashtra University, Rajkot, Gujarat 360005, India Nootan Pharmacy College, Visnagar, Gujarat 384315, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 February 2015 Received in revised form 13 May 2015 Accepted 18 May 2015 Available online 19 May 2015
The purpose of the present investigation was to improve solubility and dissolution properties of poorly water soluble drug, Embelin, a herbal active ingredient by preparing nanosuspensions. The wet media milling technique was employed for the preparation of nanosuspensions. Further, nanosuspensions were freeze dried to generate nanocrystals. Rotatable central composite design was adopted to study the effects of independent variables viz. amount of stabilizer (Pluronic F68) and amount of milling agents (Zirconium beads) on dependent variables, particle size and % drug release at 30 min. Relationship between dependent and independent variables were further investigated by multiple linear regression analysis. A significant increase was found in the solubility and dissolution rate of the formulations. Differential scanning calorimetry and Powder X-ray diffraction studies confirmed decrease in drug crystallinity. Surface electron microscopy and Transmission electron microscopy revealed plate like morphology. Results suggested remarkable improvement in the dissolution properties of Embelin by preparing nanocrystals. © 2015 Elsevier B.V. All rights reserved.
Keywords: Embelin Nanosuspension Nanocrystal Dissolution enhancement Central composite design Characterization studies
1. Introduction The poor aqueous solubility of drugs resulting in poor oral bioavailability has always been a challenging problem in pharmaceutical research. Solubility and gastrointestinal permeability are the major rate limiting steps for bioavailability and absorption [1]. Akin to many discovered chemical entities, isolated herbal active ingredients show poor aqueous solubility [2]. Till date, many formulation approaches such as salt formation, use of surfactants, use of prodrugs etc were utilized to solve the problem of solubility. Over last ten years, nanoparticle engineering has emerged for pharmaceutical applications [3]. Nanosuspensions have been widely used to deal with the problems associated with poor dissolution properties and erratic bioavailability. Nanoparticles with size ranging from 200 to 500 nm increase the saturation solubility, dissolution rate and probably the mucoadhesion of drug which might further improve the oral bioavailability [4]. In addition, nanocrystals present merits such as high drug loading, enhanced stability and less toxicity in comparison to other nanoparticles [5]. Nanosuspension formulation of some of the drugs are
* Corresponding author. E-mail address: [email protected] (K. Parmar). http://dx.doi.org/10.1016/j.jddst.2015.05.011 1773-2247/© 2015 Elsevier B.V. All rights reserved.
already in market instanced by Tricor® (fenofibrate), Cesamet® (nailone) [6]. Embelin (ELN) (Fig. 1) is a benzoquinone found in the fruits of Vidanga. ELN shows wide range of medicinal properties such as antibacterial, antifertility and antioxidant [7e9]. ELN is also studied for its antihyperglycemic effect in alloxan induced diabetes and its cytotoxic effect [10,11]. ELN have been studied for its hepatic antioxidant activity, free radical scavenging activity and lipid peroxidation in albino rats [12]. Despite of such medicinal activities of ELN, it suffers from dissolution rate limited bioavailability [13]. With this objective the present study was to formulate ELN nanocrystals with the aim to improve its solubility and dissolution properties which might further improve its oral bioavailability. Quality by Design (QbD) is a systematic approach to design, development and delivery of any pharmaceutical product or process with predefined product specifications [14]. QbD comprehends the application tools such as: design of experiments (DoE), risk assessment and process analytical technology for pharmaceutical development [15]. Central composite design (CCD) is one of the efficient designs to study the effect of different independent variables on dependent variables of formulation. Regression analysis using ANOVA is performed to enlighten the interactions between different variables to determine the optimum formulation.
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K. Parmar et al. / Journal of Drug Delivery Science and Technology 29 (2015) 1e7
Table 1. The design consists of total 9 experimental runs which included 4 factorial points, 4 star points and 1 centre point and analysed by the statistical software package Design Expert® 8.0.7.1 (Stat-Ease Inc., USA). The batch size (5 ml), drug concentration (2% w/v), Pluronic F68 as stabilizer, milling agent (zirconium beads) and solvent system (water) were kept constant in the experimental trials. Fig. 1. Chemical structure of Embelin.
2.3. Characterization of ELN nanocrystals 2. Materials and methods 2.1. Materials ELN was a gift sample from BR Nahta Pharmacy College, Mandsaur, India. Pluronic F68 was gift sample from Torrent Pharmaceuticals ltd, Ahmedabad, India. Mannitol was purchased from SD Fine Chem, Mumbai, India. Zirconium beads were obtained from Unigenetics Pvt Ltd, Delhi, India. Double distilled water was used throughout the study. 2.2. Methods 2.2.1. Preparation of nanocrystals Nanosuspensions of ELN were prepared by media milling method using water as media. Zirconium beads were used as a milling agent and Pluronic F68 was used as stabilizer. Nanosuspensions were prepared using different concentrations of stabilizer in distilled water and stirred on a magnetic stirrer (Remi, Mumbai, India) at 1000 rpm for 16 h at room temperature. The prepared suspension was then centrifuged at 5000 rpm for 10 min, supernatant was removed and the solid sediment was freeze dried using 3% w/v Mannitol as cryoprotectant (Hetro Dry Winner, Denmark). 2.2.2. Formulation and optimization of nanosuspensions using central composite design A rotatable central composite design (CCD) was selected to investigate the effects on critical quality attributes of nanosuspension. Amount of stabilizer and amount of zirconium beads were identified as critical formulation parameters and the factor levels were suitably coded. Particle sizes in nm and % drug release in 30 min (CDR30) were taken as the response variables. Independent factors and their levels used in this study are shown in
2.3.1. Particle size determination Particle size, polydispersity index and zeta potential were determined by photon correlation spectroscopy using Malvern Zetasizer (Malvern Instruments, UK). All data presented are the average values of three samples. Samples were suitably diluted with de-ionized water before analysis. 2.3.2. In vitro dissolution studies In vitro drug release studies of ELN nanocrystals and pure drug were carried out in USP type II dissolution apparatus (Model Disso 2000, Lab India) using 900 mL of phosphate buffer (pH 7.4) as dissolution media with conditions of 50 rpm and temperature 37 ± 0.5 C. Aliquots of 5 ml was withdrawn at suitable time interval (5, 10, 15, 20, 30 45, 60 and 90 min), filtered using Whatman filter paper (0.22 m), appropriately diluted and then analysed spectrophotometrically at lmax 291 nm by UV/Visible spectrophotometer (UV-1800 Shimadzu, Japan). 2.4. Characterization of optimized ELN nanocrystals 2.4.1. Saturation solubility studies Solubility studies were carried out in phosphate buffer pH 7.4. Excess of ELN and ELN nanocrystals were added to 5 ml of solvent and the mixtures were shaken for 48 h at 37 C. Suspensions were centrifuged at 5000 rpm for 10 min, supernatant was removed and thereby analysed spectrophotmetrically at lmax 291 nm after appropriate dilutions for drug concentration. 2.4.2. Differential scanning calorimetry (DSC) DSC analysis was carried out using differential scanning calorimeter (Shimadzu, DSC 60 TSW 60, Japan). Samples (ELN and optimized formulation) were accurately weighed and placed in aluminium pan and closed with a lid. Study was carried out
Table 1 Factors investigated using central composite design. Independent variables
Levels
X1 ¼ Amount of Stabilizer (Pluronic F68) (%w/v) X2 ¼ Amount of milling agent (Zirconium beads) (gm)
-a
1
0
þ1
þa
0.08 1.2
0.10 2
0.15 4
0.20 6
0.22 6.8
Table 2 Effect of independent variables on particle size and drug release at 30 min. Run
X1 (amount of stabilizer, %w/v)
X2 (amount of milling agent, gm)
Y1 (particle size, nm)
EN1 EN2 EN3 EN4 EN5 EN6 EN7 EN8 EN9
1 þ1 1 þ1 1.41 þ1.41 0 0 0
1 1 þ1 þ1 0 0 1.41 þ1.41 0
304.64 307.11 265.27 267.19 297.99 274.93 332.06 237.15 252.28
± ± ± ± ± ± ± ± ±
3.17 5.66 3.54 6.47 7.46 5.40 11.10 3.41 4.16
Y2 (% cumulative drug release at 30 min) 65.55 68.10 82.24 84.67 61.92 80.76 63.82 92.37 88.68
± ± ± ± ± ± ± ± ±
0.88 1.49 1.93 2.78 3.01 2.35 1.93 0.86 1.29
K. Parmar et al. / Journal of Drug Delivery Science and Technology 29 (2015) 1e7
grid stained with 1% w/v phosphotungstic acid solution and observed.
Table 3 Polydispersity index and zeta potential results. Batch
Polydispersity index (PI)
EN1 EN2 EN3 EN4 EN5 EN6 EN7 EN8 EN9
0.14 0.12 0.18 0.13 0.14 0.13 0.18 0.15 0.13
± ± ± ± ± ± ± ± ±
Zeta potential (z) mV 27.98 28.04 29.38 30.46 28.83 30.13 29.37 28.49 29.48
0.01 0.02 0.02 0.03 0.02 0.02 0.01 0.01 0.03
3
± ± ± ± ± ± ± ± ±
2.4.6. Stability studies of optimized ELN nanocrystals Stability studies of optimized batch of ELN nanocrystals were carried out at 40 C/75 %RH for a period of six months. Aliquots of the sample were periodically withdrawn, diluted and analysed for particle size, polydispersity index, zeta potential, saturation solubility and % drug release at 30 min (CDR30). Each sample was analysed in triplicate.
2.23 1.73 0.82 1.09 1.47 1.52 1.06 1.18 0.69
3. Results and discussion between range of 50e200 C, at a scanning rate of 10 C/min. A sealed empty pan was used as a reference. 2.4.3. Powder X-ray diffraction studies (PXRD) Polymorphic change in nanocrystal formulation was assessed by powder X-ray diffractometer (Phillips X-Pert MPD, The Netherlands) using a voltage of 45 kV, generator current 40 mA, scan step time 9 sec1 and scan step size of 0.008 (2q). The scanning rate employed was maintained over the interval of 1e50 2q1. The X-ray diffractograms of ELN and nanocrystals were recorded. 2.4.4. Scanning electron microscopy (SEM) Particle morphology was investigated using a Hitachi (S-4700, Japan) scanning electron microscope with an acceleration voltage of 30 kV. The samples were mounted on aluminium mount, sputter-coated with 9 nm of gold/palladium and imaged using scanning electron microscope. 2.4.5. Transmission electron microscopy (TEM) For visual observation, optimized batch of nanocrystals were subjected to transmission electron microscopy (TEM) (H-7000, Hitachi, Japan). Briefly, a drop of dispersion was placed on a copper
3.1. Characterization of ELN nanocrystals 3.1.1. Particle size distribution and zeta potential analysis Particle size of the formulations as shown in Table 2 ranges from 237.89 ± 0.68 nm to 332.78 ± 0.79 nm. Polydispersity index (PI) and Zeta potential values are given in Table 3. PI is the measure of globule size homogeneity. Value closer to zero, more homogeneous are the particles. PI values ranges from 0.012 ± 0.02 to 0.17 ± 0.02. Zeta potential values of EN1 to EN9 as depicted in Table 3 were found in the range of 30.46 ± 1.09 mV to 27.98 ± 2.22 mV. Zeta potential values in the range of 25 mV to 30 mV in either charge signifies a stable formulation [16]. PI and Zeta potential of optimised batch of nanocrystals were 0.13 ± 0.02 and 30.34 ± 1.06 mV respectively. 3.1.2. In vitro dissolution studies Fig. 2 shows the dissolution profiles of ELN and ELN nanocrystals in phosphate buffer pH 7.4. ELN nanocrystals exhibited significant enhancement in dissolution rate of pure drug. Increase in dissolution rate can be explained by NoyeseWhitney equation, where the dissolution rate is inversely proportional to particle size [17]. Also, hydrophobicity of drug might have decreased due to enhanced
Fig. 2. In vitro drug release studies.
Table 4 Results of regression analysis. Response
b0
b1
b2
b3
b4
b5
b6
b7
R2
Y1 Y2
252.28 88.68
8.15 6.66
33.56 10.09
0.14 0.030
17.22 8.56
16.29 5.19
13.73 1.78
9.25 5.42
0.9999 0.9997
4
K. Parmar et al. / Journal of Drug Delivery Science and Technology 29 (2015) 1e7
Fig. 3. Interaction plots; (a) Response surface plot of Y1, (b) Response surface plot of Y2 and (c) Overlay plot showing optimized batch.
K. Parmar et al. / Journal of Drug Delivery Science and Technology 29 (2015) 1e7
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Table 5 Results of ANNOVA. Response Y1 Regression FM RM Error FM RM Response Y2 Regression FM RM Error FM RM
Df (1,1)
SS
MS
F
P value
7 6
7348.20 7348.12
1049.74 1224.69
1941.09 3973.51
0.0175 0.0003
1 2 Df (2,1)
0.54 0.62 SS
0.54 0.31 MS
F
P value
7 5
1081.50 1075.17
154.50 215.03
432.76 96.43
0.0370 0.0016
1 3
0.36 6.69
0.36 2.23
Ftab ¼ 161.45 Fcal ¼ 0.148
Ftab ¼ 199.50 Fcal ¼ 8.792
Fig. 5. Differential scanning calorimetry spectra of (a) ELN and (b) OEN.
wettability and solubility due to surface stabilizers used. 3.1.3. Optimization of nanocrystals Central composite design with 2 independent variables at 3 different levels with a value 1.414 was used to study the effect on dependent variables. A total of 9 formulations were prepared as per the experimental design and characterized further for responses like particle size (nm) and % cumulative drug release at 30 min (CDR30) as shown in Table 2. The data obtained from experimental runs were subjected to regression analysis. For all the 9 batches dependent variables, particle size (Y1) and CDR30 (Y2) demonstrated wide variations from 237.15 ± 3.41 to 332.06 ± 11.10 nm and 61.92 ± 3.01 to 92.37 ± 0.86 (%) respectively indicating good influence of independent variables (X1 and X2) on the selected responses. The fitted polynomial equations for full and reduced model unfolding the response Y1 and Y2 to the transformed factors are shown in Table 4. The coefficients of the polynomials fitted well to the data, with values of R2, 0.9999 and 0.9997 for Y1 and Y2 respectively. The relationship between the dependent and independent variables was further enlightened using response surface plot shown in Fig. 3. Fig. 3a depicts an interaction effect between amount of stabilizer and amount of beads on the particle size as dependent variable. With increasing the concentration of Pluronic F68 and zirconium beads, particle size was found to be decrease. It was observed that at low and high level of stabilizer particle size was increased, this might be due to agglomeration at low
Fig. 4. Saturation solubility studies.
concentration and Ostwald ripening effect at higher concentration [18]. Moreover, increase in the concentration of stabilizer leads to increase in the viscosity of the system resulting into hindrance to the movement of milling agent which might be responsible for increase in the particle size at higher level of X1 [19]. Fig. 3b shows the response surface plot characterizing increase in the % CDR30 with increase in the concentration of stabilizer and milling agent. However, there was a decrease in % CDR30 at both high and low levels of stabilizer. This could be explicated through an increase in the particle size at these levels. Maximum % CDR30 of 92.37 ± 0.86% was found with EN8. This might be credited to decrease in particle size [20]. The ANOVA results are shown in Table 5. The F calculated value 0.148 and 8.792 is less than the table value 161.45 and 199.50 for Y1 and Y2 respectively. Using software optimisation process and response surface plots shown in Fig. 3c, level selected for X1 and X2 were 0.14 and 5.44 respectively, which gives theoretical values of 237.51 nm and 93.03% for particle size and % CDR30 respectively. Fresh formulation was prepared using the optimum levels of independent variables. The observed values of particle size and % CDR30 were found to be 237.96 ± 2.74 nm and 92.53 ± 1.92%, respectively, which were in close agreement with the theoretical values. 3.1.4. Saturation solubility studies Fig. 4 presents the saturation solubility data of ELN and ELN nanocrystals in phosphate buffer, pH 7.4. The saturation solubility of ELN nanocrystals were significantly enhanced in phosphate buffer when compared to pure ELN. This might be due to decrease in the particle size thereby increasing the dissolution pressure due to strong curvature of the nanoparticles [21]. A considerable enhancement in saturation solubility of ELN in formulation was
Fig. 6. Powder X-ray diffraction spectra of (a) ELN and (b) OEN.
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K. Parmar et al. / Journal of Drug Delivery Science and Technology 29 (2015) 1e7
Fig. 7. Scanning electron micrographs of (a) ELN and (b) OEN.
DSC studies. Fig. 6 exhibited sharp peaks of ELN at 13.3 , 16.2 , 19.0 , 21.6 , 23.5 and 25.3 demonstrating its crystalline nature. The characteristic peaks were retained in the PXRD spectra of OEN, with reduced intensities. This could be explained as the presence of added excipients influenced the peak intensities through interactions with the drug [24]. 3.1.7. Surface morphology The surface morphology graphs of ELN and OEN are presented in Fig. 7. ELN showed crystalline nature. After milling there was a considerable change in shape and surface morphology of drug. ELN exhibited plate shape crystals whereas EON appeared with irregular shape particles. The morphology of OEN was further investigated by means of TEM. Fig. 8 shows irregular nanoparticles.
Fig. 8. Transmission electron micrographs of OEN.
3.1.8. Accelerated stability studies Results of different parameters like particle size, polydispersity index, zeta potential, saturation solubility and %CDR30 of OEN evaluated at specific intervals (0, 1, 2, 3 and 6 months) are depicted in Table 6. Results showed that there was no significant change in these parameters.
observed due to the wetting action of surface stabilizer. 4. Conclusion 3.1.5. Differential scanning calorimetry studies (DSC) Fig. 5 shows DSC thermograms of pure ELN and optimized ELN nanocrystals (OEN). The thermogram of ELN showed a single sharp peak at 149 C attributing to its crystalline nature. Thermogram of OEN showed retention of characteristic peak of ELN. The endothermic peak was less sharp as compared to pure drug demonstrating a minor loss of crystallinity which might be attributed to drug interaction with the used excipients [22]. The endotherm of OEN was observed at lower temperature 147.5 C which might be attributed to the reduced particle size or miscibility of drug with excipients [23].
In the present investigation, nanocrystal formulation was proposed to enhance the dissolution properties of poorly water soluble herbal active ingredient, ELN. Significant enhancement was observed in the solubility and dissolution properties of ELN. DSC and PXRD studies indicated minor reduction in drug crystallinity. ELN nanocrystals were found to be chemically and physically stable. Thus, it can be inferred that physicochemically stable ELN nanocrystals have potential to enhance the dissolution properties of poorly water soluble drug, ELN. Conflict of interest
3.1.6. Powder X-ray diffraction studies (PXRD) PXRD studies were carried out to verify the results obtained in
The authors declare that they have no competing interest.
Table 6 Characterization of OEN after Accelerated stability studies at 40 C/75%RH. Sample time (month)
Particle size (nm)
0 1 2 3 6
237.96 237.45 238.14 238.29 238.04
± ± ± ± ±
2.73 1.94 2.38 2.44 2.25
Polydispersity index 0.13 0.13 0.14 0.14 0.14
± ± ± ± ±
0.02 0.01 0.01 0.02 0.01
Zeta potential z (mV)
Saturation solubility (mg/ml)
30.34 30.28 29.48 30.15 29.45
8113.79 8060.06 8123.89 8043.06 8096.04
± ± ± ± ±
1.06 1.67 0.91 0.98 1.01
± ± ± ± ±
104.64 93.01 120.65 70.88 158.12
% CDR30 (min) 92.98 92.86 92.67 92.24 92.75
± ± ± ± ±
0.85 1.06 0.43 0.93 0.71
K. Parmar et al. / Journal of Drug Delivery Science and Technology 29 (2015) 1e7
Acknowledgement The authors are thankful to Mr. Swapnil Goyal (Asst. Prof., BR Nahta College of Pharmacy, Mandsaur) for providing the gift sample of ELN. The support of Torrent Pharmaceuticals Ltd, Ahmedabad is gratefully acknowledged to provide the gift sample of Poloxamer. References [1] L. Dandan, X. Heming, T. Baocheng, Y. Kun, P. Hao, M. Shilin, Y. Xinggang, P. Weisan, Fabrication of carvedilol nanosuspensions through the anti-solvent precipitation-ultrasonication method for the improvement of dissolution rate and oral bioavailability, AAPS PharmSciTech. 13 (1) (2013) 295e304. [2] R. Ravinchandra, Studies on dissolution behaviour of nanoparticulate curcumin formulation, Adv. Nanoparticles 2 (2013) 51e59. [3] S. Verma, E. Gokhale, D.J. Burgessa, A comparative study of top down and bottom up approaches for the preparation of micro/nanosuspensions, Int. J. Pharm. 380 (2009) 261e322. [4] C. Jacobs, R.H. Müller, Production and characterization of a budesonide nanosuspension for pulmonary administration, Pharm. Res. 19 (2002) 189e194. [5] S.C. Kim, D.W. Kim, Y.H. Shim, J.S. Bang, H.S. Oh, S.W. Kim, M.H. Seo, In vivo evaluation of polymeric micellar paclitaxel formulation: toxicity and efficacy, J. Control. Release 72 (2001) 191e202. [6] A.H. Junghanns Jens-Uwe, R.H. Müller, Nanocrystal technology: drug delivery and clinical applications, Int. J. Nanomed 3 (2008) 295e310. [7] M. Chitra, C.S. Shyamala Devi, E. Sukumar, Antibacterial activity of embelin, Fitoterapia 74 (4) (2003) 401e403. [8] N. Radhakrishnan, M. Alam, Antifertility effects of embelin in albino rats, Indian J. Exp. Biol. 13 (1) (1975) 70e71. [9] R. Gupta, A.K. Sharma, M.C. Sharma, R.S. Gupta, Antioxidant activity and protection of pancreatic b-cells by embelin in streptozotocin-induced diabetes, J. Diabetes 4 (3) (2012) 248e256. [10] S.N. Tripathi, Screening of hypoglycemic action in certain indigenous drugs, J. Res. Ind. Med. 14 (1979) 159e169. [11] B. Joy, S. Lakshmi, Antiproliferative properties of embelia ribes, Open Proc. Chem. J. 3 (2010) 17e22.
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[12] R. Gupta, A.K. Sharma, M.C. Sharma, R.S. Gupta, Antioxidant activity and protection of pancreatic b-cells by embelin in streptozotocin-induced diabetes, J. Diabetes 4 (3) (2012) 248e256. [13] R.A. Pathan, U. Bhandari, Preparation and characterization of embelinphospholipid complex as effective drug delivery tool, J. Incl. Phenom. Macro. Chem. 69 (2012) 139e147. [14] X.Y. Lawrence, Pharmaceutical quality by design: product and process development, understanding, and control, Pharm. Res. 25 (2008) 781e791. [15] S. Verma, Y. Lan, R. Gokhale, D.J. Burgess, Quality by design approach to understand the process of nanosuspension preparation, Int. J. Pharm. 377 (2009) 185e198. [16] F. Shakeel, N. Haq, M. El-Badry, F.K. Alanazi, I.A. Alsarra, Ultra fine super selfnanoemulsifying drug delivery system (SNEDDS) enhanced solubility and dissolution of indomethacin, J. Mol. Liq. 180 (2013) 89e94. € m, The effect of particle size and shape on the surface [17] M. Mosharraf, C. Nystro specific dissolution rate of microsized practically insoluble drugs, Int. J. Pharm. 122 (1995) 35e47. [18] V.M. Pandya, J.K. Patel, D.J. Patel, Formulations and optimization of nanosuspensions for enhancing simvastatin dissolution using central composite design, Dissolution Technol. (2011) 40e45. [19] J. Deng, L. Huang, F. Liu, Understanding the structure and stability of paclitaxel nanocrystals, Int. J. Pharm. 390 (2010) 242e249. [20] K.K. Singh, K.K. Srinivasan, K. Gowthamarajan, D.S. Singare, D. Prakash, N.B. Gaikwad, Investigation of preparation parameters of nanosuspension by top-down media milling to improve the dissolution of poorly water-soluble glyburide, Eur. J. Pharm. Biopharm. 78 (2011) 441e446. € hler and [21] A.K. Shchekin, A.I. Rusanov, Generalization of the GibbseKelvineKo OstwaldeFreundlich equations for a liquid film on a soluble nanoparticle, J.Chem. Phys. 129 (2008) 154116. [22] S.M.A. Hany, Y. Peter, M.A.A. Ahmed, B. Nicholas, Hydrocortisone nanosuspensions for ophthalmic delivery: a comparative study between microfluidic nanoprecipitation and wet milling, J. Control. Release 149 (2011) 175e181. [23] C.L. Jackson, G.B. McKenna, The melting behaviour of organic materials confined in porous solids, J. Chem. Phys. 93 (1990) 9002e9011. [24] S. Sugimoto, T. Niwa, Y. Nakanishi, K. Danjo, Novel ultra-cryo milling and cogrinding technique in liquid nitrogen to produce dissolution-enhanced nanoparticles for poorly water-soluble drugs, Chem. Pharm. Bull. 60 (2012) 325e333.
Contents lists available at ScienceDirect
Journal of Drug Delivery Science and Technology journal homepage: www.elsevier.com/locate/jddst
Original research
Formulation and optimization of Embelin nanosuspensions using central composite design for dissolution enhancement Komal Parmar a, *, Jayvadan Patel b, Navin Sheth a a b
Department of Pharmaceutical Sciences, Saurashtra University, Rajkot, Gujarat 360005, India Nootan Pharmacy College, Visnagar, Gujarat 384315, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 February 2015 Received in revised form 13 May 2015 Accepted 18 May 2015 Available online 19 May 2015
The purpose of the present investigation was to improve solubility and dissolution properties of poorly water soluble drug, Embelin, a herbal active ingredient by preparing nanosuspensions. The wet media milling technique was employed for the preparation of nanosuspensions. Further, nanosuspensions were freeze dried to generate nanocrystals. Rotatable central composite design was adopted to study the effects of independent variables viz. amount of stabilizer (Pluronic F68) and amount of milling agents (Zirconium beads) on dependent variables, particle size and % drug release at 30 min. Relationship between dependent and independent variables were further investigated by multiple linear regression analysis. A significant increase was found in the solubility and dissolution rate of the formulations. Differential scanning calorimetry and Powder X-ray diffraction studies confirmed decrease in drug crystallinity. Surface electron microscopy and Transmission electron microscopy revealed plate like morphology. Results suggested remarkable improvement in the dissolution properties of Embelin by preparing nanocrystals. © 2015 Elsevier B.V. All rights reserved.
Keywords: Embelin Nanosuspension Nanocrystal Dissolution enhancement Central composite design Characterization studies
1. Introduction The poor aqueous solubility of drugs resulting in poor oral bioavailability has always been a challenging problem in pharmaceutical research. Solubility and gastrointestinal permeability are the major rate limiting steps for bioavailability and absorption [1]. Akin to many discovered chemical entities, isolated herbal active ingredients show poor aqueous solubility [2]. Till date, many formulation approaches such as salt formation, use of surfactants, use of prodrugs etc were utilized to solve the problem of solubility. Over last ten years, nanoparticle engineering has emerged for pharmaceutical applications [3]. Nanosuspensions have been widely used to deal with the problems associated with poor dissolution properties and erratic bioavailability. Nanoparticles with size ranging from 200 to 500 nm increase the saturation solubility, dissolution rate and probably the mucoadhesion of drug which might further improve the oral bioavailability [4]. In addition, nanocrystals present merits such as high drug loading, enhanced stability and less toxicity in comparison to other nanoparticles [5]. Nanosuspension formulation of some of the drugs are
* Corresponding author. E-mail address: [email protected] (K. Parmar). http://dx.doi.org/10.1016/j.jddst.2015.05.011 1773-2247/© 2015 Elsevier B.V. All rights reserved.
already in market instanced by Tricor® (fenofibrate), Cesamet® (nailone) [6]. Embelin (ELN) (Fig. 1) is a benzoquinone found in the fruits of Vidanga. ELN shows wide range of medicinal properties such as antibacterial, antifertility and antioxidant [7e9]. ELN is also studied for its antihyperglycemic effect in alloxan induced diabetes and its cytotoxic effect [10,11]. ELN have been studied for its hepatic antioxidant activity, free radical scavenging activity and lipid peroxidation in albino rats [12]. Despite of such medicinal activities of ELN, it suffers from dissolution rate limited bioavailability [13]. With this objective the present study was to formulate ELN nanocrystals with the aim to improve its solubility and dissolution properties which might further improve its oral bioavailability. Quality by Design (QbD) is a systematic approach to design, development and delivery of any pharmaceutical product or process with predefined product specifications [14]. QbD comprehends the application tools such as: design of experiments (DoE), risk assessment and process analytical technology for pharmaceutical development [15]. Central composite design (CCD) is one of the efficient designs to study the effect of different independent variables on dependent variables of formulation. Regression analysis using ANOVA is performed to enlighten the interactions between different variables to determine the optimum formulation.
2
K. Parmar et al. / Journal of Drug Delivery Science and Technology 29 (2015) 1e7
Table 1. The design consists of total 9 experimental runs which included 4 factorial points, 4 star points and 1 centre point and analysed by the statistical software package Design Expert® 8.0.7.1 (Stat-Ease Inc., USA). The batch size (5 ml), drug concentration (2% w/v), Pluronic F68 as stabilizer, milling agent (zirconium beads) and solvent system (water) were kept constant in the experimental trials. Fig. 1. Chemical structure of Embelin.
2.3. Characterization of ELN nanocrystals 2. Materials and methods 2.1. Materials ELN was a gift sample from BR Nahta Pharmacy College, Mandsaur, India. Pluronic F68 was gift sample from Torrent Pharmaceuticals ltd, Ahmedabad, India. Mannitol was purchased from SD Fine Chem, Mumbai, India. Zirconium beads were obtained from Unigenetics Pvt Ltd, Delhi, India. Double distilled water was used throughout the study. 2.2. Methods 2.2.1. Preparation of nanocrystals Nanosuspensions of ELN were prepared by media milling method using water as media. Zirconium beads were used as a milling agent and Pluronic F68 was used as stabilizer. Nanosuspensions were prepared using different concentrations of stabilizer in distilled water and stirred on a magnetic stirrer (Remi, Mumbai, India) at 1000 rpm for 16 h at room temperature. The prepared suspension was then centrifuged at 5000 rpm for 10 min, supernatant was removed and the solid sediment was freeze dried using 3% w/v Mannitol as cryoprotectant (Hetro Dry Winner, Denmark). 2.2.2. Formulation and optimization of nanosuspensions using central composite design A rotatable central composite design (CCD) was selected to investigate the effects on critical quality attributes of nanosuspension. Amount of stabilizer and amount of zirconium beads were identified as critical formulation parameters and the factor levels were suitably coded. Particle sizes in nm and % drug release in 30 min (CDR30) were taken as the response variables. Independent factors and their levels used in this study are shown in
2.3.1. Particle size determination Particle size, polydispersity index and zeta potential were determined by photon correlation spectroscopy using Malvern Zetasizer (Malvern Instruments, UK). All data presented are the average values of three samples. Samples were suitably diluted with de-ionized water before analysis. 2.3.2. In vitro dissolution studies In vitro drug release studies of ELN nanocrystals and pure drug were carried out in USP type II dissolution apparatus (Model Disso 2000, Lab India) using 900 mL of phosphate buffer (pH 7.4) as dissolution media with conditions of 50 rpm and temperature 37 ± 0.5 C. Aliquots of 5 ml was withdrawn at suitable time interval (5, 10, 15, 20, 30 45, 60 and 90 min), filtered using Whatman filter paper (0.22 m), appropriately diluted and then analysed spectrophotometrically at lmax 291 nm by UV/Visible spectrophotometer (UV-1800 Shimadzu, Japan). 2.4. Characterization of optimized ELN nanocrystals 2.4.1. Saturation solubility studies Solubility studies were carried out in phosphate buffer pH 7.4. Excess of ELN and ELN nanocrystals were added to 5 ml of solvent and the mixtures were shaken for 48 h at 37 C. Suspensions were centrifuged at 5000 rpm for 10 min, supernatant was removed and thereby analysed spectrophotmetrically at lmax 291 nm after appropriate dilutions for drug concentration. 2.4.2. Differential scanning calorimetry (DSC) DSC analysis was carried out using differential scanning calorimeter (Shimadzu, DSC 60 TSW 60, Japan). Samples (ELN and optimized formulation) were accurately weighed and placed in aluminium pan and closed with a lid. Study was carried out
Table 1 Factors investigated using central composite design. Independent variables
Levels
X1 ¼ Amount of Stabilizer (Pluronic F68) (%w/v) X2 ¼ Amount of milling agent (Zirconium beads) (gm)
-a
1
0
þ1
þa
0.08 1.2
0.10 2
0.15 4
0.20 6
0.22 6.8
Table 2 Effect of independent variables on particle size and drug release at 30 min. Run
X1 (amount of stabilizer, %w/v)
X2 (amount of milling agent, gm)
Y1 (particle size, nm)
EN1 EN2 EN3 EN4 EN5 EN6 EN7 EN8 EN9
1 þ1 1 þ1 1.41 þ1.41 0 0 0
1 1 þ1 þ1 0 0 1.41 þ1.41 0
304.64 307.11 265.27 267.19 297.99 274.93 332.06 237.15 252.28
± ± ± ± ± ± ± ± ±
3.17 5.66 3.54 6.47 7.46 5.40 11.10 3.41 4.16
Y2 (% cumulative drug release at 30 min) 65.55 68.10 82.24 84.67 61.92 80.76 63.82 92.37 88.68
± ± ± ± ± ± ± ± ±
0.88 1.49 1.93 2.78 3.01 2.35 1.93 0.86 1.29
K. Parmar et al. / Journal of Drug Delivery Science and Technology 29 (2015) 1e7
grid stained with 1% w/v phosphotungstic acid solution and observed.
Table 3 Polydispersity index and zeta potential results. Batch
Polydispersity index (PI)
EN1 EN2 EN3 EN4 EN5 EN6 EN7 EN8 EN9
0.14 0.12 0.18 0.13 0.14 0.13 0.18 0.15 0.13
± ± ± ± ± ± ± ± ±
Zeta potential (z) mV 27.98 28.04 29.38 30.46 28.83 30.13 29.37 28.49 29.48
0.01 0.02 0.02 0.03 0.02 0.02 0.01 0.01 0.03
3
± ± ± ± ± ± ± ± ±
2.4.6. Stability studies of optimized ELN nanocrystals Stability studies of optimized batch of ELN nanocrystals were carried out at 40 C/75 %RH for a period of six months. Aliquots of the sample were periodically withdrawn, diluted and analysed for particle size, polydispersity index, zeta potential, saturation solubility and % drug release at 30 min (CDR30). Each sample was analysed in triplicate.
2.23 1.73 0.82 1.09 1.47 1.52 1.06 1.18 0.69
3. Results and discussion between range of 50e200 C, at a scanning rate of 10 C/min. A sealed empty pan was used as a reference. 2.4.3. Powder X-ray diffraction studies (PXRD) Polymorphic change in nanocrystal formulation was assessed by powder X-ray diffractometer (Phillips X-Pert MPD, The Netherlands) using a voltage of 45 kV, generator current 40 mA, scan step time 9 sec1 and scan step size of 0.008 (2q). The scanning rate employed was maintained over the interval of 1e50 2q1. The X-ray diffractograms of ELN and nanocrystals were recorded. 2.4.4. Scanning electron microscopy (SEM) Particle morphology was investigated using a Hitachi (S-4700, Japan) scanning electron microscope with an acceleration voltage of 30 kV. The samples were mounted on aluminium mount, sputter-coated with 9 nm of gold/palladium and imaged using scanning electron microscope. 2.4.5. Transmission electron microscopy (TEM) For visual observation, optimized batch of nanocrystals were subjected to transmission electron microscopy (TEM) (H-7000, Hitachi, Japan). Briefly, a drop of dispersion was placed on a copper
3.1. Characterization of ELN nanocrystals 3.1.1. Particle size distribution and zeta potential analysis Particle size of the formulations as shown in Table 2 ranges from 237.89 ± 0.68 nm to 332.78 ± 0.79 nm. Polydispersity index (PI) and Zeta potential values are given in Table 3. PI is the measure of globule size homogeneity. Value closer to zero, more homogeneous are the particles. PI values ranges from 0.012 ± 0.02 to 0.17 ± 0.02. Zeta potential values of EN1 to EN9 as depicted in Table 3 were found in the range of 30.46 ± 1.09 mV to 27.98 ± 2.22 mV. Zeta potential values in the range of 25 mV to 30 mV in either charge signifies a stable formulation [16]. PI and Zeta potential of optimised batch of nanocrystals were 0.13 ± 0.02 and 30.34 ± 1.06 mV respectively. 3.1.2. In vitro dissolution studies Fig. 2 shows the dissolution profiles of ELN and ELN nanocrystals in phosphate buffer pH 7.4. ELN nanocrystals exhibited significant enhancement in dissolution rate of pure drug. Increase in dissolution rate can be explained by NoyeseWhitney equation, where the dissolution rate is inversely proportional to particle size [17]. Also, hydrophobicity of drug might have decreased due to enhanced
Fig. 2. In vitro drug release studies.
Table 4 Results of regression analysis. Response
b0
b1
b2
b3
b4
b5
b6
b7
R2
Y1 Y2
252.28 88.68
8.15 6.66
33.56 10.09
0.14 0.030
17.22 8.56
16.29 5.19
13.73 1.78
9.25 5.42
0.9999 0.9997
4
K. Parmar et al. / Journal of Drug Delivery Science and Technology 29 (2015) 1e7
Fig. 3. Interaction plots; (a) Response surface plot of Y1, (b) Response surface plot of Y2 and (c) Overlay plot showing optimized batch.
K. Parmar et al. / Journal of Drug Delivery Science and Technology 29 (2015) 1e7
5
Table 5 Results of ANNOVA. Response Y1 Regression FM RM Error FM RM Response Y2 Regression FM RM Error FM RM
Df (1,1)
SS
MS
F
P value
7 6
7348.20 7348.12
1049.74 1224.69
1941.09 3973.51
0.0175 0.0003
1 2 Df (2,1)
0.54 0.62 SS
0.54 0.31 MS
F
P value
7 5
1081.50 1075.17
154.50 215.03
432.76 96.43
0.0370 0.0016
1 3
0.36 6.69
0.36 2.23
Ftab ¼ 161.45 Fcal ¼ 0.148
Ftab ¼ 199.50 Fcal ¼ 8.792
Fig. 5. Differential scanning calorimetry spectra of (a) ELN and (b) OEN.
wettability and solubility due to surface stabilizers used. 3.1.3. Optimization of nanocrystals Central composite design with 2 independent variables at 3 different levels with a value 1.414 was used to study the effect on dependent variables. A total of 9 formulations were prepared as per the experimental design and characterized further for responses like particle size (nm) and % cumulative drug release at 30 min (CDR30) as shown in Table 2. The data obtained from experimental runs were subjected to regression analysis. For all the 9 batches dependent variables, particle size (Y1) and CDR30 (Y2) demonstrated wide variations from 237.15 ± 3.41 to 332.06 ± 11.10 nm and 61.92 ± 3.01 to 92.37 ± 0.86 (%) respectively indicating good influence of independent variables (X1 and X2) on the selected responses. The fitted polynomial equations for full and reduced model unfolding the response Y1 and Y2 to the transformed factors are shown in Table 4. The coefficients of the polynomials fitted well to the data, with values of R2, 0.9999 and 0.9997 for Y1 and Y2 respectively. The relationship between the dependent and independent variables was further enlightened using response surface plot shown in Fig. 3. Fig. 3a depicts an interaction effect between amount of stabilizer and amount of beads on the particle size as dependent variable. With increasing the concentration of Pluronic F68 and zirconium beads, particle size was found to be decrease. It was observed that at low and high level of stabilizer particle size was increased, this might be due to agglomeration at low
Fig. 4. Saturation solubility studies.
concentration and Ostwald ripening effect at higher concentration [18]. Moreover, increase in the concentration of stabilizer leads to increase in the viscosity of the system resulting into hindrance to the movement of milling agent which might be responsible for increase in the particle size at higher level of X1 [19]. Fig. 3b shows the response surface plot characterizing increase in the % CDR30 with increase in the concentration of stabilizer and milling agent. However, there was a decrease in % CDR30 at both high and low levels of stabilizer. This could be explicated through an increase in the particle size at these levels. Maximum % CDR30 of 92.37 ± 0.86% was found with EN8. This might be credited to decrease in particle size [20]. The ANOVA results are shown in Table 5. The F calculated value 0.148 and 8.792 is less than the table value 161.45 and 199.50 for Y1 and Y2 respectively. Using software optimisation process and response surface plots shown in Fig. 3c, level selected for X1 and X2 were 0.14 and 5.44 respectively, which gives theoretical values of 237.51 nm and 93.03% for particle size and % CDR30 respectively. Fresh formulation was prepared using the optimum levels of independent variables. The observed values of particle size and % CDR30 were found to be 237.96 ± 2.74 nm and 92.53 ± 1.92%, respectively, which were in close agreement with the theoretical values. 3.1.4. Saturation solubility studies Fig. 4 presents the saturation solubility data of ELN and ELN nanocrystals in phosphate buffer, pH 7.4. The saturation solubility of ELN nanocrystals were significantly enhanced in phosphate buffer when compared to pure ELN. This might be due to decrease in the particle size thereby increasing the dissolution pressure due to strong curvature of the nanoparticles [21]. A considerable enhancement in saturation solubility of ELN in formulation was
Fig. 6. Powder X-ray diffraction spectra of (a) ELN and (b) OEN.
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K. Parmar et al. / Journal of Drug Delivery Science and Technology 29 (2015) 1e7
Fig. 7. Scanning electron micrographs of (a) ELN and (b) OEN.
DSC studies. Fig. 6 exhibited sharp peaks of ELN at 13.3 , 16.2 , 19.0 , 21.6 , 23.5 and 25.3 demonstrating its crystalline nature. The characteristic peaks were retained in the PXRD spectra of OEN, with reduced intensities. This could be explained as the presence of added excipients influenced the peak intensities through interactions with the drug [24]. 3.1.7. Surface morphology The surface morphology graphs of ELN and OEN are presented in Fig. 7. ELN showed crystalline nature. After milling there was a considerable change in shape and surface morphology of drug. ELN exhibited plate shape crystals whereas EON appeared with irregular shape particles. The morphology of OEN was further investigated by means of TEM. Fig. 8 shows irregular nanoparticles.
Fig. 8. Transmission electron micrographs of OEN.
3.1.8. Accelerated stability studies Results of different parameters like particle size, polydispersity index, zeta potential, saturation solubility and %CDR30 of OEN evaluated at specific intervals (0, 1, 2, 3 and 6 months) are depicted in Table 6. Results showed that there was no significant change in these parameters.
observed due to the wetting action of surface stabilizer. 4. Conclusion 3.1.5. Differential scanning calorimetry studies (DSC) Fig. 5 shows DSC thermograms of pure ELN and optimized ELN nanocrystals (OEN). The thermogram of ELN showed a single sharp peak at 149 C attributing to its crystalline nature. Thermogram of OEN showed retention of characteristic peak of ELN. The endothermic peak was less sharp as compared to pure drug demonstrating a minor loss of crystallinity which might be attributed to drug interaction with the used excipients [22]. The endotherm of OEN was observed at lower temperature 147.5 C which might be attributed to the reduced particle size or miscibility of drug with excipients [23].
In the present investigation, nanocrystal formulation was proposed to enhance the dissolution properties of poorly water soluble herbal active ingredient, ELN. Significant enhancement was observed in the solubility and dissolution properties of ELN. DSC and PXRD studies indicated minor reduction in drug crystallinity. ELN nanocrystals were found to be chemically and physically stable. Thus, it can be inferred that physicochemically stable ELN nanocrystals have potential to enhance the dissolution properties of poorly water soluble drug, ELN. Conflict of interest
3.1.6. Powder X-ray diffraction studies (PXRD) PXRD studies were carried out to verify the results obtained in
The authors declare that they have no competing interest.
Table 6 Characterization of OEN after Accelerated stability studies at 40 C/75%RH. Sample time (month)
Particle size (nm)
0 1 2 3 6
237.96 237.45 238.14 238.29 238.04
± ± ± ± ±
2.73 1.94 2.38 2.44 2.25
Polydispersity index 0.13 0.13 0.14 0.14 0.14
± ± ± ± ±
0.02 0.01 0.01 0.02 0.01
Zeta potential z (mV)
Saturation solubility (mg/ml)
30.34 30.28 29.48 30.15 29.45
8113.79 8060.06 8123.89 8043.06 8096.04
± ± ± ± ±
1.06 1.67 0.91 0.98 1.01
± ± ± ± ±
104.64 93.01 120.65 70.88 158.12
% CDR30 (min) 92.98 92.86 92.67 92.24 92.75
± ± ± ± ±
0.85 1.06 0.43 0.93 0.71
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