DSpace at VNU: Amorphous isradipine nanosuspension by the sonoprecipitation method

G Model IJP 14259 1–5 International Journal of Pharmaceutics xxx (2014) xxx–xxx Contents lists available at ScienceDirect International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm Pharmaceutical nanotechnology Amorphous isradipine nanosuspension by the sonoprecipitation method Q1 Thao Truong-Dinh Tran * , Phuong Ha-Lien Tran * , Minh Ngoc Uyen Nguyen, Khanh Thi My Tran, Minh Nguyet Pham, Phuc Cao Tran, Toi Van Vo Pharmaceutical Engineering Laboratory, Biomedical Engineering Department, International University, Vietnam National University, Ho Chi Minh City, Viet Nam A R T I C L E I N F O A B S T R A C T Article history: Received 31 May 2014 Received in revised form 28 July 2014 Accepted 14 August 2014 Available online xxx The aims of this study are to increase and explain the mechanism of dissolution enhancement of isradipine using the sonoprecipitation method for stable nanosuspensions There have been still few of published researches on formulation of isradipine using nanoparticle engineering Nanosuspension systems were prepared upon various factors including amplitude and the time length of ultrasonication The dissolution test was performed according to the USP paddle method in intestinal fluid (pH 6.8) The crystalline structure of drug, the molecular interaction, morphology and size of nanosuspension were also investigated to determine the mechanism of dissolution enhancement The sonoprecipitation method with use of HPMC showed its potential in enhancement of the drug release rate Stable nanosuspension was significantly depended on amplitude and time of ultrasonication since these factors affected on the size of nanoparticles The synergistic effects of reduction of drug crystallinity and particle size could increase the dissolution rate of isradipine by providing a stable nanosuspension This work may contribute to a new strategy for improvement dissolution rate of isradipine ã 2014 Published by Elsevier B.V Keywords: Nanosuspension Sonoprecipitation method Crystallinity Dissolution enhancement Q2 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 Introduction Currently, more than 40% of drugs are poorly water-soluble, leading to the poor bioavailability (Müller et al., 2006; Patel and Agrawal, 2011) Therefore, one of the major current challenges of the pharmaceutical industry is related to strategies that improve the water solubility of drugs A number of studies have been conducted with the aim to enhance solubility and dissolution rate of poorly water-soluble drugs Sonoprecipitation method is one of the promising approaches for formulation of poorly water-soluble drug compounds (Dalvi and Dave, 2009, 2010; Dhumal et al., 2008; Liu et al., 2012; Miao et al., 2011; Moorthi and Kathiresan, 2013; Zheng et al., 2010) because ultrasound has been proved to be an effective method for breaking down particles into nanoparticles (Kim et al., 2013) Factors such as amplitude level and ultrasonication time can be controlled to produce highly stable nanosuspension and high drug dissolution rate Isradipine (IS), is a dihydropyridine calcium channel blocker, was chosen as the model drug in this research It is virtually * Corresponding authors Tel.: +84 37244270x3328; fax: +84 37244271 E-mail addresses: ttdthao@hcmiu.edu.vn (T.T.-D Tran), thlphuong@hcmiu.edu.vn (P.H.-L Tran) insoluble in water but freely soluble in acetone (Chrysant and Cohen, 1997; Leroueil-Le Verger et al., 1998) and may be degraded under the light An IS formulation with improvement of dissolution and photo-instability hence should be investigated In previous studies, we have successfully enhanced the dissolution rate and controlled release rate of IS using solid dispersion techniques (Tran and Tran, 2013; Tran et al., 2010) Recently, Park et al has developed the inclusion complex of IS and b-cyclodextrin for improvement of photo-instability and dissolution profile (Park et al., 2013) However, there have been few of such studies on nano-sized formulation of IS apart from the research of Verger et al (Leroueil-Le Verger et al., 1998) The aim of those nanoparticles was to prolong the antihypertensive effect of the drug Herein, this study was aimed to develop nanoparticulate systems for improving bioavailability of IS through an improved drug dissolution Various polymers and ultrasonication conditions were used to investigate the improvement of dissolution rate and nanosuspention stablity The structural behaviors of drug were characterized by powder X-ray diffraction (PXRD) Morphology and particle size analyses were also conducted through transmission electron microscope (TEM) and scanning electron microscope (SEM) The potential molecular interaction between drug and polymer was also investigated by Fourier transform infrared spectroscopy (FTIR) http://dx.doi.org/10.1016/j.ijpharm.2014.08.017 0378-5173/ ã 2014 Published by Elsevier B.V Please cite this article in press as: Tran, T.T.-D., et al., Amorphous isradipine nanosuspension by the sonoprecipitation method Int J Pharmaceut (2014), http://dx.doi.org/10.1016/j.ijpharm.2014.08.017 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 G Model IJP 14259 1–5 T.T.-D Tran et al / International Journal of Pharmaceutics xxx (2014) xxx–xxx increments of 0.02 from 5 to 60 (diffraction angle 2-theta) at s/step, using a zero background sample holder Materials and methods 51 2.1 Materials 52 60 Polyethylene oxide N-60K (PEO), hydroxypropyl methylcellulose cps (HPMC 6) and hydroxypropyl methylcellulose 4000 cps were provided by from Dow Chemical Company (Midland, Michigan, USA) Acetone was purchased from Xilong Chemical Co., Ltd., (Shantou, Guangdong, China) The solvents (methanol and acetonitrile) for high performance liquid chromatography (HPLC) were purchased from Fisher Scientific (Pittsburgh, Pennsylvania, USA) All other chemicals were of analytical grade and were used without further purification 61 2.2 Methods 62 2.2.1 HPLC analysis HPLC system (Dionex, USA) with Luna m C18 analytical column (150 mm  4.6 mm) was used to determine IS concentration The UV detector was set at 325 nm to analyze the column effluent The mixture of methanol, deionized water, and acetonitrile (7:3:5) was used as a mobile phase The entire solution was filtered through a 0.45 mM membrane filter and was degassed prior to use 20 mL of samples were injected into HPLC system for analysis The standard solutions were constructed in the range of 0.05–10 ppm for calibration with good linearity (R2 = 0.9991) 53 54 55 56 57 58 59 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 Q3 93 94 2.2.2 Preparation of IS nanosuspensions by sonoprecipitation method Firstly, IS was dissolved in acetone (30 mg/mL) Meanwhile, polymer was dissolved in water to obtain an anti-solvent (1 mg/mL) Then, the organic solution was quickly introduced into the anti-solvent under stirring After the anti-solvent precipitation, the samples were immediately treated with ultrasonic liquid processors (QSONICA, USA) at different amplitudes and time Acetone was completely evaporated in the oven at 40  C overnight To prepare samples for physicochemical analyses, the nanosuspension was lyophilized at À50  C until formation of powder The detailed formulations are described in Table 2.2.3 Dissolution studies The samples equivalent to mg IS were exposed to 900 mL enzyme-free simulated intestinal fluid (pH 6.8) for 120 using the USP apparatus II (50 rpm, 37  C) Samples were withdrawn at predetermined intervals and replaced with an equivalent amount of fresh medium to maintain a constant dissolution volume The concentrations of IS were finally analyzed by HPLC as described above 2.2.4 Powder X-ray diffraction (PXRD) The freeze-dried sample, IS, HPMC analyzed the crystallinity by X-ray diffractometer (Bruker D8 Advance, Germany) using Cu-Ka radiation at a voltage of 40 kV, 50 mA The samples were scanned in Table Formulation of IS suspensions Formulation IS HPMC (mg) (mg) F1 F2 F3 F4 F5 F6 F7 F8 5 5 5 5 30 – – 20 10 5 HPMC 4000 (mg) 30 – – – – – – PEO N-60K (mg) Sonication amplitude (level) Sonication time (min) – 30 – – – – – 5 5 5 2 5 5 5 2.2.5 Fourier transform infrared spectroscopy (FTIR) A FTIR spectrophotometer (Bruker Vertex 70, Germany) was used to investigate the spectra of IS, HPMC and freeze-dried sample The wavelength was scanned from 500 to 4000 cmÀ1 with a resolution of cmÀ1 KBr pellets were prepared by gently mixing mg of the sample with 200 mg KBr 95 96 97 Q4 98 99 100 101 102 2.2.6 Transmission electron microscopy and scanning electron microscopy Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) were used to characterize surface morphology and particle shape The samples were examined using JEM-1400 Transmission Electron Microscope (Jeol, Japan) and JSM-6480LV (Jeol, Japan) 103 Results and discussion 110 3.1 Effect of polymer types and polymer concentrations on dissolution rate of IS 111 In order to find out the possibility of the varied release rate of drug by polymers, three types of polymers (HPMC 6, HPMC 4000 and PEO) were used to prepare the nanosuspension under the same ultrasonication conditions (Fig 1) All of the polymers significantly increased the dissolution rate of IS as compared to the dissolution rate of pure drug Thus, the sonoprecipitation method was effective in enhancing the drug release rate However, drug release from the nanosuspension of PEO formulation showed the slowest as compared to that from the nanosuspension of HPMC 4000 and HPMC These results demonstrated that type of polymers is an important factor for increasing dissolution rate of drug by sonoprecipitation method To evaluate the effect of polymer concentrations and optimize the sufficient amount of polymer used in sonoprecipitation method, HPMC was chosen and prepared with drug and polymer at ratio 1:5 (F4), 1:2 (F5) and 1:1 (F6) Fig shows that the dissolution rate of IS was insignificantly changed by increasing amount of the polymer Therefore, the ratio 1:1 between HPMC and IS was selected as the optimum for investigation of ultrasonication conditions 113 120 100 Drug release [%] 50 80 60 40 F3 (PEO) F1 (HPMC 6) F2 (HPMC 4000) pure IS 20 20 40 60 80 100 120 Time [min] Fig Effect of polymer types (PEO, HPMC or HPMC 4000) on dissolution rate of IS at pH 6.8 Please cite this article in press as: Tran, T.T.-D., et al., Amorphous isradipine nanosuspension by the sonoprecipitation method Int J Pharmaceut (2014), http://dx.doi.org/10.1016/j.ijpharm.2014.08.017 104 105 106 107 108 109 112 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 G Model IJP 14259 1–5 T.T.-D Tran et al / International Journal of Pharmaceutics xxx (2014) xxx–xxx 120 Drug release [%] 100 80 60 F4 (20 mg HPMC 6) F5 (10 mg HPMC 6) F6 (5 mg HPMC 6) 40 20 20 40 60 80 100 3.2 Effect of ultrasonication conditions and optimization of formulations 132 In order to investigate the effect of amplitude of ultrasonication on drug dissolution rate as well as the stability of suspensions, the amplitude for ultrasonication was decreased to level for (F7) Similarly, the ultrasonication time was also reduced to (F8) to investigate the dissolution rate Figs and show dissolution profiles of drug from the suspensions which were prepared by various amplitudes and time Drug release from all of the formulations reached at about 100% after 120 regardless of amplitude level or ultrasinication time Ultrasonication has been used to reduce the particle size and enhance drug dissolution High intensity sonic waves provide energy that cause mechanical stress and direct heat to break intermolecular interactions Moreover, the 134 120 Time [min] Fig Effect of HPMC concentrations on dissolution rate of IS at pH 6.8 120 Drug release [%] 100 80 F7 (amplitude 2) F6 (amplitude 5) 60 40 20 20 40 60 80 100 120 Time [min] Fig Effect of ultrasonication amplitudes on dissolution rate of IS at pH 6.8 120 Drug release [%] 100 80 60 F8 (4 mins) F7 (5 mins) 40 20 20 40 60 80 100 120 Timne [min] Fig Effect of ultrasonication time on dissolution rate of IS at pH 6.8 Fig Precipitation observation of formulation F6, F7, F8 at room temperature until h Please cite this article in press as: Tran, T.T.-D., et al., Amorphous isradipine nanosuspension by the sonoprecipitation method Int J Pharmaceut (2014), http://dx.doi.org/10.1016/j.ijpharm.2014.08.017 133 135 136 137 138 139 140 141 142 143 144 145 G Model IJP 14259 1–5 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 T.T.-D Tran et al / International Journal of Pharmaceutics xxx (2014) xxx–xxx intensity of sonic waves is directly proportional to the amplitude of vibration of the ultrasonic source An acceleration of the amplitude may lead to an increase in the intensity of vibration and an increase in the effect of chemical reaction (Santos et al., 2008) In addition to the investigation of ultrasonication conditions, the precipitation of samples was also observed after sample preparation for optimization Fig illustrates the precipitation state of the F6, F7, and F8 until h Sedimentation in the samples prepared at the amplitude level appeared more heavily than that in the sample prepared at the amplitude level The phenomenon may be a consequence of particle size, drug concentration, inherent density difference between carrier and nanoparticle drug, etc Sedimentation is one of the significant factors which Q5 determine a long-term stability of nanosuspension (Che et al., 2012) The amplitude and time under ultrasonication had significant impacts on the stability of suspensions With low input amplitude and time, the particles settled down faster than in the case of the samples prepared by higher input amplitude and longer time Higher input amplitude may cause higher destructiveness to solid particles in preparation of a dispersion (Hielscher, 2005) Size of the particles depends on energy per volume, where energy is the product of power input and exposure time (Hielscher, 2005) When the suspensions are well-prepared, they exhibit strong stability, indicating no sedimentation for a determined period Thus, the drug-loaded particles would prolong its suspension state to promote its effective act for treatment Among the samples, the one prepared at the amplitude level in (F6) showed the best stability 3.3 Mechanism of dissolution enhancement Particle size and morphology of the pure IS and IS nanosuspension were studied by SEM and TEM Fig 6A shows that the pure IS powder has irregular shapes with particle size generally larger (5–50 mm) and had different morphology However, the size of IS particles was reduced to 0.5–1 mm when IS was encapsulated in HPMC under ultrasonication conditions of level of amplitude for (Fig 6B) Specially, the size of particles was around 50 nm under ultrasonication conditions of level of amplitude for (Fig 6C) The IS nanosuspension was more uniform as compared to the pure IDP The differences of morphological properties may affect wettability and hence, inducing or hindering drug dissolution rate These results also indicated that the increase of amplitude and time caused the reduced particle size, and hence, resulting in the improvement of nanosuspension dissolution and stability (Kakran et al., 2010; Liu et al., 2010, 2012) The X-ray diffraction was performed on pure IS, HPMC and F6 formulation to investigate the effect of sonoprecipitation method on the crystallinity of IS through HPMC 6-based formulation Fig shows that the PXRD pattern of HPMC had a broad characteristic peak between 16 and 24 of 2-theta Meanwhile, pure IS exhibited numerous characteristic peaks from 10 to 30 of 2-theta, indicating that the drug was at the highly crystalline form which may lead to poorly water-soluble property of IS (Tran et al., 2010) However, a number of IS characteristic peaks decreased or disappeared in the case of IS encapsulation in the HPMC nanoparticles Specifically, the peaks at 11.5, 11.7, 12.2, 13.9, 17.5, 19.3 and 23 of IS in nanosuspension were decreased in intensity as compared to the pure IS The other peaks of IS were almost disappeared in nanosuspension formulation A large reduction in characteristic peaks indicated an amorphous state of IS (Hu et al., 2003; Tran et al., 2010) These results clearly elucidated the roles of HPMC and sonoprecipitation process in transforming the crystalline structure of IS to partially crystalline form, leading to the enhanced drug dissolution rate (Tran et al., 2009; Vasconcelos et al., 2007) FT-IR spectra of the F6 formulation, HPMC and pure IS were characterized to investigate if there was any interaction between Fig SEM image of pure IS (A) and TEM images of particles in formulations (B) F7; (C) F6 Please cite this article in press as: Tran, T.T.-D., et al., Amorphous isradipine nanosuspension by the sonoprecipitation method Int J Pharmaceut (2014), http://dx.doi.org/10.1016/j.ijpharm.2014.08.017 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 G Model IJP 14259 1–5 T.T.-D Tran et al / International Journal of Pharmaceutics xxx (2014) xxx–xxx form may attribute the main mechanism to the dissolution enhancement The stability and size of nanosuspension were significantly affected by ultrasonication conditions such as amplitude level and time F6 (amplitude level in min) was an optimized formulation to obtain high drug dissolution rate and stable nanosuspension for further studies in therapeutic applications 229 Acknowledgement 235 This research is funded by the International University, VNU- Q6 HCM under grant number SV-06-2012/HÐ-ÐHQT-QHQT&QLKH 236 References 238 230 231 232 233 234 Pure IS HPMC F6 10 20 30 40 50 2-Theta Fig PXRD patterns of pure IS, HPMC and freeze-dried sample of F6 formulation HPMC F6 pure IS 4000 3000 2000 1000 Wavelength (cm-1) Fig FTIR spectra of pure IS, HPMC and freeze-dried sample of F6 formulation 212 223 drug molecule and HPMC that may affect the dissolution rate of drug (Fig 8) The broad peak from 3100 to 3700 cmÀ1 in the spectrum F6 was attributed to the ÀÀOH groups which obviously appeared in the spectrum of F6 formulation, indicating the presence of HPMC However, there was an addition of a peak at 3346 cmÀ1 in this broad spectrum This peak was attributed to the absorption of amino group NÀ ÀH of IS (Tran and Tran, 2013) Besides, other peaks of IS were also presented on the spectrum of F6 formulation The results demonstrated that there was no interaction between HPMC and IS Therefore, the dissolution enhancement of IS in nanosuspension was not caused by any interaction between IS and HPMC 224 Conclusion 225 The sonoprecipitation method showed that it was an effective method for preparation of stable 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method Int J Pharmaceut (2014), http://dx.doi.org/10.1016/j.ijpharm.2014.08.017 ... preparation for optimization Fig illustrates the precipitation state of the F6, F7, and F8 until h Sedimentation in the samples prepared at the amplitude level appeared more heavily than that in the. .. Thus, the sonoprecipitation method was effective in enhancing the drug release rate However, drug release from the nanosuspension of PEO formulation showed the slowest as compared to that from the. .. the nanosuspension of HPMC 4000 and HPMC These results demonstrated that type of polymers is an important factor for increasing dissolution rate of drug by sonoprecipitation method To evaluate the