Controlling the size and morphology of griseofulvin

J Nanopart Res (2015)17:256 DOI 10.1007/s11051-015-3066-6

RESEARCH PAPER

Controlling the size and morphology of griseofulvin nanoparticles using polymeric stabilizers by evaporationassisted solvent–antisolvent interaction method Raj Kumar . Prem Felix Siril

Received: 30 March 2015 / Accepted: 3 June 2015 Ó Springer Science+Business Media Dordrecht 2015

Abstract Griseofulvin (GF) is a potential drug for cancer therapy. However, its application is limited by its poor water solubility. Ultrafine GF nanoparticles were prepared through evaporation-assisted solvent– antisolvent interaction method for improving its solubility. Acetone was used as the solvent and water was used as the antisolvent. It was observed that particle size could be controlled by varying the concentration of GF in acetone. Average particle size was very low, 16 ± 4 and 28 ± 8 nm, when the concentration of GF was 5 and 25 mM, respectively, in acetone. However, the particle size increased drastically to more than 3 lm, when the concentration was increased to 50 mM. Interestingly, the presence of optimized concentration of polyvinylpyrrolidone (PVP) and hydroxypropyl methylcellulose (HPMC) as stabilizers in the antisolvent resulted in significant reduction of particle size. Particle size decreased to less than 40 nm in the presence of the polymeric stabilizers, even when the concentration was 50 mM.

Field emission scanning electron microscopy, transmission electron microscopy, and atomic force microscopy imaging revealed that the polymeric stabilizers encapsulated very small GF particles and thus stabilized them. The solubility of GF-HPMC, GFPVP, and the bare GF particles that were prepared from 50 mM solution (micro-GF) was nearly 24, 19, and 11 times, respectively, higher than that of raw-GF. In vitro dissolution studies revealed that almost 100 % of the drug was released in 60 min from GF-PVP and GF-HPMC. Fourier transform infrared spectroscopy did not detect any strong interaction between GF and the stabilizers. X-ray diffraction showed that the prepared GF nanoparticles and the micro-GF were in polymorphic form I. Differential scanning calorimetric studies showed that the crystallinity of the nanoformulated GF was only slightly lower than that of raw-GF. Thus, particle size reduction and the presence of stabilizers led to significant enhancement in solubility and dissolution rate.

Electronic supplementary material The online version of this article (doi:10.1007/s11051-015-3066-6) contains supplementary material, which is available to authorized users.

Keywords Drug nanoparticles Griseofulvin Antisolvent precipitation Solubility Bioavailability Polymeric stabilizer Nanomedicine

R. Kumar P. F. Siril (&) School of Basic Sciences and Advanced Material Research Centre, Indian Institute of Technology Mandi, Mandi 175005, Himachal Pradesh, India e-mail: [email protected] R. Kumar e-mail: [email protected]

Introduction Griseofulvin (GF, ((2S,60 R)-7-chloro-20 ,4,6-trimethoxy-60 -methyl-3H,40 H-spiro[1-benzofurane-2,10 cyclohex[2]ene]-3,40 -dione)) is an antibiotic and

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antifungal drug (El-Nakeeb et al. 1965). It is a commonly used drug for the treatment of fungal infections of the skin and nails (Sulzberger and Baer 1959). Recently, GF gained medical attention due to its ability to disrupt mitotic spindles and act as potential inhibitor of centrosomal clustering in tumor cells (Panda et al. 2005). It has been shown that GF in combination with nocodazole enhances the effect of nocodazole against the colon cancer cells in vivo (Wang et al. 2002). GF is a highly hydrophobic drug having low solubility in water. Poor water solubility of drugs in general leads to the low bioavailability and variable and incomplete absorption through the gastrointestinal tract after oral administration (Dahan et al. 2009). Thus, poor solubility of drugs in water is a big challenge for pharmaceutical industry (MeriskoLiversidge et al. 2003). About 40 % of the newly synthesized drug molecules are having poor water solubility and hence low bioavailability (Kipp 2004). Drug molecules having high permeability and low solubility are classified as class II active pharmaceutical ingredients (APIs) in biopharmaceutical classification system (BCS) (Kasim et al. 2004). GF is a BCS class II drug. Bioavailability of BCS class II APIs is limited by their solubility and dissolution rate. Hence, their bioavailability can be enhanced by increasing the solubility and dissolution rate. There are a number of ways by which bioavailability of drugs having poor water solubility can be enhanced (Chen et al. 2011). Particle size reduction usually leads to enhancement of solubility due to the enhancement in surface area. Micronization or nanosizing of drug particles has been proven to be a tangible option for achieving enhanced solubility (Kesisoglou et al. 2007). Formulating drugs with water-soluble polymers, surfactants, lipids, and other excipients can also lead to enhanced solubility (Kumar and Randhawa 2013). Nano-sized particles of drugs can be produced using a number of methods. Different techniques for the preparation of nanoparticles can be broadly classified into two categories based on the production principle: bottom-up and top-down processes (Chen et al. 2011; Mijatovic et al. 2005). The top-down methods start with larger solid particles and break them down into nano/microparticles mechanically. The wet and jet milling and homogenization using rotor stator or high pressure homogenization are the examples for the extensively studied top-down methods (Abbas et al. 2013; Jinno et al. 2008; Midoux et al.

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1999; Nykamp et al. 2002; Schultz et al. 2004; Yuan et al. 2008). In bottom-up methods, fine particles are produced by precipitation from a solution. These methods give better control over particle properties such as size, morphology, and crystal nature as compared to that of top-down methods (Desai et al. 2012; Verma et al. 2009). Antisolvent precipitation is a simple and convenient method for the preparation of nano-sized drug particles. Finding a suitable solvent to dissolve the API and a suitable antisolvent that has good miscibility with the solvent is the key challenge in the bottom-up ‘‘antisolvent precipitation’’ processes. The antisolvent precipitation method was developed nearly 20 years ago. Nakanishi used antisolvent precipitation method for the preparation of organic nanoparticles (Kasai et al. 2012). Later on, this method was extensively used for the nanoformulation due to the advantages such as simplicity, cost-effectiveness, and ease in scaling up (Hu et al. 2011). Typically, a solution of an API is injected into an antisolvent under mixing. The solvent must be the one which gives better solubility of the API and must be miscible with the antisolvent. Mostly water is used as the antisolvent. Organic solvents may also be used as the antisolvent. If a suitable solvent–antisolvent pair can be identified, the antisolvent precipitation process is a simple and effective technique to produce nano-sized particles. For BCS class II APIs, it is ideal to have water as the antisolvent. It is necessary to decrease mixing and increase precipitation process in antisolvent precipitation methods to produce nanoparticles with smaller particle size and narrow size distribution (Thorat and Dalvi 2012). We have recently developed the evaporation-assisted solvent–antisolvent interaction (EASAI) method where the rate of precipitation is enhanced using conditions favoring evaporation of the solvent (Kumar and Siril 2014). In the EASAI method, the solution of drug in a low-boiling organic solvent is injected to a hot antisolvent. The temperature of the antisolvent is kept above the boiling point of the solvent so that the rate of precipitation is enhanced by the evaporation of the solvent. Here, we report the preparation of GF nanoparticles with particle size below 40 nm and a spherical morphology using EASAI method. It was found that encapsulation of drug nanoparticles using a water-soluble polymer led to even higher solubility and dissolution rate (Kumar and Siril 2014). Hence, two water-soluble polymers,


polyvinylpyrrolidone (PVP) and hydroxypropyl methylcellulose (HPMC), were used to increase the dissolution of GF. HPMC and PVP are widely used as stabilizers for the controlled drug release (Kumar and Siril 2014). Both the polymers are easily accepted; it is easy to formulate the necessary doses and these are approved by the U.S. Food and Drug Administration (FDA).

Materials and methods Materials GF, acetone, ethanol, HPMC (avg. MW 86,000), PVP (avg. MW 40,000), and sodium dodecyl sulfate (SDS) were purchased from Sigma-Aldrich and were used as received. HPLC micro-syringe was purchased from Hamilton, USA. Syringe filters with pore sizes 0.22–0.45 lm and Whatmann Anodisc 25 filter of 20 nm pore size with filtration setup were purchased from Millipore, USA. Ultra-pure water (18.2 MX cm) from double stage water purifier (ELGA PURELAB Option R7) was used for all the experiments. Preparation of GF nanoparticles Accurately weighed drug was dissolved in an appropriate volume of acetone to prepare stock solutions of 50 mM. Typically, 8.81 g of GF was dissolved in 500 ml acetone. Solutions of lower concentrations (5 and 25 mM) were prepared by diluting the stock solution. All the prepared drug solutions were filtered using syringe filter of pore size 0.22 lm to remove any particles present in it. The antisolvent (25 ml) was heated to 70 °C in a 100-ml conical flask. The drug solution (100 ll) of known concentration was quickly injected to the hot antisolvent using a micro-syringe under magnetic stirring to precipitate nanoparticles. Stirring was continued up to few minutes after injection. Aqueous solution of the respective stabilizers was used as antisolvent in the preparation of GFPVP and GF-HPMC nanoparticles. Particle size and morphology Particle size (z-average diameter, d/nm), polydispersity index (PDI), and zeta potential of the precipitated nanoparticles were measured using dynamic light

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scattering (DLS) method (Zetasizer Nano ZS, Malvern Instrument Ltd., UK) at 25 °C. The instrument contains a 4 mW He–Ne laser operating at a wavelength of 633 nm and incorporates noninvasive backscatter optics (NIBS). All measurements were made at a detection angle of 173°. Particle size and morphology of the samples were characterized by field emission scanning electron microscopy (FESEM), transmission electron microscopy (TEM), and atomic force microscopy (AFM) imaging. Aqueous suspension of the nanoparticles was drop casted on clean glass slides and dried. The dried glass slide was transferred onto a carbon tape applied on a clean SEM stub. Samples were subsequently sputter coated with gold at 20 mA for 180 s before the FESEM (Supra55 VP model, ZIESS Instruments Pvt. Ltd.) observations were carried out. The particle size was measured manually from FESEM images using Image J software. The TEM imaging of the prepared GF-PVP and GF-HPMC nanoparticles were done after negative staining. The aqueous suspensions of the samples were drop coated on TEM grids and incubated for 180 s. The samples were then stained using an aqueous solution of 2 % (w/v) phosphotungstic acid (PTA). Excess PTA was removed by washing with ultra-pure water. Imaging was done using TEM (FEI, Tecnai G2, and S-Twin) at 200 kV. AFM (AFM Dimension ICON, Bruker Pvt. Ltd.) imaging of samples was done in tapping mode with a scanning rate of 0.9 Hz and TESPA tip. Samples were dispersed in water and drop coated on a silicon wafer for imaging. Drug encapsulation efficiency Quantification of the encapsulated drug was done using a Shimadzu double beam UV–Visible spectrophotometer. A calibration curve was prepared by measuring the absorbance of a series of GF solutions in ethanol in the concentration range of 2–20 lM. The encapsulation efficiency (EE) was calculated by measuring the amount of drug in the GF-PVP and GF-HPMC nanoparticles. Known quantities of GFPVP and GF-HPMC were dissolved in 3 ml ethanol. The dispersion was filtered through Whatman Anodisc 25 filter having pore size 20 nm to remove any particles present in it. Absorption spectrum of the solution was recorded and the concentration of the drug was found out from the calibration curve. The %

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EE was calculated using the following formula (Song et al. 2008): % EE ¼

Weight of GF released into ethanol 100 Weight of GF added initially

Solubility and in vitro dissolution studies Solubility of the prepared nanoparticles and the raw drug was measured using a Shimadzu double beam UV– Visible spectrophotometer. Typically, a known quantity of each sample (20 mg) was weighed accurately and added to a 100-ml conical flask containing 5 ml of water. The aqueous suspension was magnetically stirred inside a thermostatic water bath at 37 ± 1 °C for 24 h at 100 rpm. After 24 h, the suspension was centrifuged and the supernatant was filtered using Whatman Anodisc 25 filter of pore size 20 nm to remove undissolved particles. The solution was further diluted to the required concentration by adding water. The concentration of drug in the solution was determined using optical density at 296 nm wavelength. In vitro drug dissolution tests were performed using a USP Microprocessor Dissolution Test Apparatus, Model 1912, (Electronics India). Typically, 80 mg of sample was added to 900 ml of water containing 0.27 % (w/v) SDS (Bhakay et al. 2013). The suspension was stirred at 100 rpm at 37 ± 1 °C. Aliquots of 2 ml release medium were withdrawn at different time intervals. After each withdrawal, the corresponding volume (2 ml) was replenished with fresh medium to maintain sink condition (Bhakay et al. 2013). The withdrawn aliquots were filtered using Whatman Anodisc 25 filter of pore size 20 nm and the absorption spectra were recorded. The quantity of drug dissolved was obtained from the calibration curve. FTIR spectroscopy Fourier transform infrared spectroscopy (FTIR) spectroscopy was performed using the Perkin Elmer FTIR emission spectrometer (Spectrum Two). The FTIR spectrum of the samples was recorded in the frequency range of 4000–600 cm-1 with a resolution of 4 cm-1 and eight scans. The samples were properly grounded with KBr powder and then pressed to obtain a suitably sized pellet for FTIR spectrum measurement. Pure KBr pellet was used for background correction.

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XRD analysis X-ray diffraction (XRD) measurements were performed on a Smart Lab X-Ray Diffractometer (Rigaku, Japan) using Cu Ka radiation as X-ray source (k = 0.15418 nm) at room temperature. The voltage and current applied were 45 kV and 100 mA, respectively. Samples were placed in a glass sample holder and scanned in the 2h range of 10°–70° with scan rate of 2°/min and step size of 0.02°. DSC analysis Differential scanning calorimetric (DSC) analysis was carried out using a Netzsch STA 449 F1 Jupiter instrument. The samples (2-3 mg) were taken in an alumina crucible with a lid having a pin hole in the middle and heated from room temperature to 500 °C at a heating rate of 5 °C/min, under N2 atmosphere with a flow rate of 60 ml/min. An empty crucible was used as the reference.

Results and discussion Acetone was selected as the solvent, due to its low boiling point and also the high solubility of drug in it. Injection of drug solution into the antisolvent under stirring led to the precipitation of particles. There was no turbidity formation, indicating the tiny size of particles and also very low concentration. The stirring was continued up to few minutes after injection to complete the mixing and to yield smaller particle size with narrow size distribution (Sinha et al. 2013). Although antisolvent precipitation is a very simple method, a number of experimental factors such as temperature of antisolvent, concentration of the solution, rate of injection, nozzle geometry, ratio of solvent to antisolvent, and ultrasonication are known to influence the particle size (Sinha et al. 2013). In a previous work, we studied the effect of different experimental parameters such as ratio of solvent to antisolvent and temperature of antisolvent on particles size and morphology of nanoparticles. Smaller particles were formed with solvent-to-antisolvent ratio of 1:250, at 70 °C (Kumar et al. 2014, 2015). High temperature (70 °C) led to quick evaporation of solvent (acetone) as its boiling point is 56 °C (Kumar et al. 2014). The temperature of antisolvent was fixed


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at 70 °C and the ratio of solvent to antisolvent at 1:250 for the present study. This is because we found that EASAI leads to smaller particle size, irrespective of the chemical nature of the solute being precipitated (Kumar and Siril 2014; Kumar et al. 2014). Similarly, the effect of ratio of solvent to antisolvent on particle size also may not change for the same pair of solvent and antisolvent. However, the effect of concentration of GF in its solution and the concentration of stabilizers (PVP and HPMC) on particles size was studied and the results are presented in the following sections. The effect of presence of stabilizer and its concentration on particle size The effect of stabilizer on particle size was studied using two different water-soluble polymers. Concentration of GF in the antisolvent was 50 mM while solvent-to-antisolvent ratio was 1:250 and the temperature of the antisolvent was 70 °C. Z-average particle size that was measured using DLS and values of PDI are reported in Table 1. Particle size was more than 3 lm when no polymer was present in the antisolvent. It is evident from Table 1 that the presence of the polymers led to considerable reduction in particle size as per DLS from [3 lm to 151 ± 40 and 132 ± 14 nm, respectively, when optimum quantities of PVP and HPMC were used as stabilizers. There was variation in particle sizes with change in concentration of the polymers also. Particle size was lowest when the concentration of PVP and HPMC in water was 0.2 and 0.1 % (w/v), respectively. Concentrations above and below this optimum concentration [0.2 and 0.1 % (w/v) for PVP and HPMC respectively)] led to an increase in particle size. Stabilizers control the particle size by getting adsorbed on the Table 1 Optimization of stabilizer concentration with 50 mM GF at 70 °C

% (w/v)

particles and thus preventing aggregation. There may not be enough polymer molecules available to form at least a monolayer over the particles at lower concentrations. This lack of availability of sufficient amount of stabilizers to control the growth the particles resulted in larger particles. At higher concentration of the polymers, increase in osmotic pressure leads to increased attraction among colloidal particles leading to agglomeration (Dalvi and Dave 2010). When the concentration of stabilizers is at an optimum, the particle growth was controlled by stabilizers resulting in smaller particles with narrow size distribution. The drastic reduction in particle size in the presence of the polymeric stabilizers also indicates that they are effective not only in preventing aggregation, but also in reducing the particle size by controlling the diffusion of GF molecules towards the nanocrystals. The GF nanoparticles prepared with the optimized stabilizer concentration were further characterized thoroughly and will be denoted as GF-PVP and GFHMPC. The bare GF particle that was prepared from the solution at 50 mM, having particle size more than 3 lm was also characterized thoroughly and will be denoted as micro-GF. Effect of drug concentration and stabilizers on particles size, zeta potential and morphology The effect of changing the concentration of the drug solution from 5 to 50 mM on the size of particles was studied. Some representative FESEM images of GF nanoparticles that were prepared from GF solution of varying concentrations are shown in Fig. 1. FESEM images of GF-PVP and GF-HPMC nanoparticles are shown in Fig. 2. The particle size from DLS measurements and from FESEM imaging are reported in Table 2. From the DLS data presented in Table 2, it is

PVP d (nm)

256

HPMC PDI

d (nm)

PDI

0.05

–

–

646 ± 261

0.64 ± 0.11

0.1

1556 ± 184

0.43 ± 0.28

132 ± 14

0.39 ± 0.18

0.15

–

–

495 ± 74

0.67 ± 0.21

0.2

151 ± 40

0.54 ± 0.19

–

–

0.3

190 ± 88

0.37 ± 0.04

–

–

0.4

1023 ± 275

0.69 ± 0.05

–

–

0.5

2606 ± 1241

0.93 ± 0.15

–

–

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Fig. 2 FESEM nanoparticles

Fig. 1 FESEM images of GF nanoparticles prepared from solutions of varying concentrations using EASAI method. Concentration of GF in acetone in each case is inscribed on the images

apparent that the particle size increases from 225 ± 21 to 252 ± 29 as the concentration of drug increases from 5 to 25 mM. The particle size increased drastically to [3 lm, when the concentration was increased to 50 mM. A similar trend of increase in the particle size with the increasing concentration of the drug was also observed from the FESEM analysis. The particle size measured from FESEM imaging increased from 16 ± 4 to 28 ± 8 nm as the concentration of drug was increased from 5 to 25 mM. The particle size increased significantly to [3 lm when

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images

of

GF-PVP

and

GF-HPMC

the concentration of drug was increased to 50 mM. Increase in the particle size with the increasing concentration of the solute was observed in our previous studies also (Kumar and Siril 2014; Kumar et al. 2014). The increase in particle size with the increasing concentration can be explained based on the increased availability of solute molecules for particle growth. Nucleation occurs at the interface between antisolvent and solvent droplets in the antisolvent precipitation process. A fixed volume of solution was injected to a fixed volume of antisolvent, while varying the concentration of GF. Hence, the number of droplets and the number of nuclei formed could be similar even when the concentration of solute is different. However, when the concentration of GF is more, there is an adequate supply of GF molecules in the solution for the prolonged growth of the nuclei to form larger particles. Z-average particle size measured by DLS was always significantly higher than the actual particle size observed in FESEM images. This may be due to the presence of a small number of large particles. Such a difference in particle size that is measured using DLS


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Table 2 Particle size measured from DLS and FESEM; PDI and zeta potential of the prepared micro-GF, GF-PVP, and GF-HPMC S. no.

Compositions

Particles size d (nm) DLS

FESEM

PDI

f

1

5 mM GF

225 ± 21

16 ± 04

0.34 ± 0.10

-19.48 ± 1.93

2

25 mM GF

252 ± 29

28 ± 08

0.29 ± 0.02

-13.12 ± 1.04 -8.15 ± 5.6

a

3

50 mM GF

[3000

3000–4500

0.45 ± 0.10

4

50 mM GF-PVP

151 ± 40

36 ± 13

0.54 ± 0.19

5

50 mM GF-HPMC

132 ± 14

28 ± 9

0.39 ± 0.18

a

-8.6 ± 1.82 -1.86 ± 2.3

Average particle size was not calculated as the particles were not spherical

and FESEM is widely reported in the literature (Dou et al. 2013). We have observed similar results previously, and the possible reasons for this are explained elsewhere (Kumar and Siril 2014; Kumar et al. 2014). Zeta potential of bare GF nanoparticles was found to be negative and the actual values varied with particle size. Smaller particles had more negative zeta potential and the values gradually became less negative with the increase in particle size. The zeta potential became less negative in the presence of PVP when compared to that of bare GF nanoparticles of smaller size. However, the zeta potential of GF-HPMC was closer to zero. Particle stabilization by the polymers is purely steric in nature. The observations made here are similar to the reported synthesis of GF nanoparticles by grinding in the presence of PVP (Pongpeerapat et al. 2004). From Fig. 1, it is evident that spherical nanoparticles with very small sizes were formed when the concentration was kept at 5 and 25 mM. But when the concentration was 50 mM, micron-sized facetted crystals with truncated edges were formed. It is evident from Fig. 2 that the morphology of GF particles was spherical in the presence of the polymers, even when the concentration was 50 mM. Thus, the polymers not only lowered the particle size, but also altered the morphology. This shows that the polymers bind on the surface of the GF nanoparticles and a monolayer was formed that prevents further growth of the smaller particles and also prevents aggregation. The GF nanoparticles that were prepared in the presence of stabilizers were characterized using TEM and AFM imaging and some representative images are presented in Figs. 3 and 4, respectively. From Fig. 3, it is clearly evident that the sizes of the particles are less

than 40 nm. The dark core surrounded by a light corona was observed for most of the particles, as shown in Fig. 3 and Figs. S1 and S2 (Electronic Supporting Information). This clearly indicates that the surface of the particles is coated with the stabilizer. A single core encapsulating more than one nanocrystal of GF was also seen sparingly in the TEM images. Spherical morphology and the very small particle size of the GF-PVP and GF-HPMC nanoparticles were also clearly evident in the AFM images given in Fig. 4.

Fig. 3 TEM images of GF-PVP and GF-HPMC nanoparticles

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Fig. 4 AFM images of GFPVP and GF-HPMC nanoparticles

Soft dark layers around the particles were observed in AFM images also due to the coating of stabilizer on the surface of GF nanoparticles. Individual particles were observed without much agglomeration in the FESEM, TEM, and AFM images. It indicates that particles do not get agglomerated even after drying and storage. Mild sonication was sufficient to redisperse the particles in water to prepare samples for FESEM, TEM, and AFM imaging. FESEM, TEM, and AFM imaging thus confirmed that the prepared nanoparticles of GF-PVP and GF-HPMC have spherical core– shell morphology and particle size below 40 nm. Particle size and the polymer coating should have profound effect on the properties such as solubility and drug release profile of the drug. Hence, after optimizing the conditions of preparing bare as well as polymer-stabilized GF nanoparticles, a thorough comparative study was conducted on the properties of the GF nanoparticles with the raw-GF. The solubility, chemical, crystallographic, and thermal characteristics of micro-GF, GF-PVP, and GF-HPMC were characterized and compared with that of the rawGF and the details are presented in the following sections. Drug encapsulation efficiency The drug was easily soluble in ethanol and hence it was used as a solvent to study the entrapment efficiency of the polymeric stabilizers used in the present study. The entrapment efficiencies for PVP and HPMC were 92 and 96 %, respectively. This result indicates that EASAI method is suitable for the

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preparation of GF nanoparticles with very high EF of drug. Solubility Solubility of the prepared samples and raw-GF in water was measured using UV–Vis spectrophotometry. The nano-GF samples as well as the micro-GF showed enhanced solubility when compared to rawGF. The polymer-supported GF nanoparticles showed higher solubility than the bare micro-GF. Solubility of the samples was in the following order: GFHPMC [ GF-PVP [ micro-GF [ raw-GF. The solubility of GF-HPMC, GF-PVP, and micro-GF was 24, 19, and 11 times, respectively, higher than the solubility of raw-GF. Enhanced solubility of nanosized GF particles compared to raw-GF is mainly due to the enhanced surface area. Increased presence of amorphous content in the particles also could be a reason for enhancement in solubility (Dhumal et al. 2008). In vitro dissolution studies The in vitro dissolution profiles for the nano-GF samples, micro-GF, and the raw-GF are shown in Fig. 5. The results revealed that there is significant increase in the rate of drug release for the prepared nano-sized samples when compared to that of microGF and raw-GF. Initial burst release was observed for all the four samples. However, the amount of drug released during the initial period was different for various samples. More GF molecules were released


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et al. 2011). As there was no peak shift for the characteristic peaks of GF, there was no evidence for hydrogen bonding (Vasanthavada et al. 2005). Hence, the interaction between the polymer and GF is of the weak van der Waals type. Crystal nature

Fig. 5 Drug release profile of the prepared samples and raw-GF

initially from the nano-sized samples when compared to that from raw-GF. Interestingly, the amount of drug released in the first 5 min for micro-GF was slightly higher than that of GF-PVP. This may be due to the presence of small amount of amorphous GF also along with micron-sized crystals of GF. After the burst release phase lasting for about 5 min, the release rate slowed down for all the samples except GF-HPMC. The rate of drug release increased again after a short induction period of about 10 min. It can be seen from Fig. 5 that 98 and 96 % of the drug was released from GF-HPMC and GF-PVP, respectively, in 60 min. However, only 60 and 32 % of the drug was released from micro-GF and raw-GF, respectively, in 60 min. Thus, HPMC was more effective in increasing the solubility and rate of dissolution than PVP. Increase in rate of drug release in nano-sized particles is mainly due to the decrease in particle size. Infrared spectroscopy FTIR spectroscopy was used to probe whether there is any change in chemical composition, inclusion of solvent, and presence of interaction between GF and the polymeric stabilizers or not. The FTIR spectra of all samples are reported in Fig. S3 (Electronic Supporting Information). The nano-sized GF samples showed similar bands to micro-GF and raw-GF. The characteristic bands of GF were observed at 1659 and 1707 cm-1 due to cyclohexenyl-ketone (C=O) and phenyl, respectively, for all the samples (Cutrignelli

XRD patterns for all the samples along with raw-GF are presented in Fig. S4 (Electronic Supporting Information). The micro-GF, GF-PVP, and GF-HPMC showed similar XRD patterns. But the patterns were different to that of the raw-GF. The XRD pattern of the prepared nano-sized samples matched well with the polymorphic form I of GF as reported in the literature (Mahieu et al. 2013). GF is reported to have at least three polymorphic forms, namely form I, form II, and form III (Mahieu et al. 2013). It appears that raw-GF contained a mixture of different polymorphs (Beck et al. 2013; Mahieu et al. 2013). As the polymorphic form of the raw-GF was different from that of the nano-sized particles, a direct comparison of crystallinity of the nanoparticles with raw-GF was not attempted. The relative crystallinity among the nano-sized samples with the micro-GF was studied based on the intensity of most intense peak at 2h, 16.6°. The intensity of the peak at 16.6° and hence the crystallinity was determined to be in the following order: GF-PVP [ GF-HPMC [ micro-GF. Apparently, micro-GF had lower crystallinity compared to GF-HPMC and GF-PVP. DSC analysis An overlay of DSC thermal curves for all samples is presented in Fig. S5 (Electronic Supporting Information). The corresponding data are summarized in Table 3. An endothermic process was recorded for all the samples at around 216 °C. This corresponds to melting of GF (Thakur and Gupta 2006). From the data, it can be concluded that the nanoformulated GF has slightly lower melting point than raw-GF. Slight lowering in melting point is generally observed for the nanoparticles due to decrease in the particle size. Relative crystallinity of the samples was also calculated from the DSC data by assuming that the raw-GF was 100 % crystalline. It was found by comparing the enthalpy of melting of the samples that the micro-GF was less crystalline than GF-PVP and GF-HPMC.

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Table 3 Differential scanning calorimetric data of raw-GF and the prepared samples Sample

T(Onset) (°C)

T(Peak) (°C)

Enthalpy (J g-1)

% Crystallinitya

Raw-GF

217.4

218.9

59.58

100

Micro-GF

217.2

218.6

33.64

56.46

GF-PVP GF-HPMC

216.2 216.9

217.8 218.2

50.99 47.67

85.58 80.01

a

Percentage crystallinity was calculated from the DSC data

Comparison of solubility and dissolution profile of GF nano and micro-particles A number of papers on the preparation using different techniques and characterization of micro/nanoparticles of GF are reported in literature. A comparison of the effect of different methods of preparation for particle size and drug release profile is given Table T1 (Electronic Supporting Information). Those papers that reported the preparation of sub-micron-sized GF particles were only included. A summary of literature reports on micronization of GF has been reported by Dalvi et al. (Dalvi and Dave 2010). It is clearly evident from Table T1 that the preparation of GF nanoparticles with particle size less than 40 nm could be accomplished by EASAI method. The smallest average particle size reported so far in the literature is more than double (85 nm) than that reported by us (Trotta et al. 2003). A direct comparison of the drug release profiles that are reported in the literature is not possible due to the use of different techniques and dissolution media. Trotta et al. and Bhakay et al. reported that 85 and 97 % of GF were released in 1 and 2 min, respectively (Bhakay et al. 2013; Trotta et al. 2003). It may be mainly because of the higher amorphous nature of the samples. Amorphous drugs tend to crystalize during storage and this may lead to change in dissolution profile. Thus, the possibility of producing ultrafine nanoparticles with low amorphous content using the EASAI method seems to be attractive when compared to other methods of nanoformulation of drugs.

Conclusions EASAI method offers a greater control on particle size of GF by the manipulation of concentration of the drug in the solvent. Ultrafine nanoparticles of GF with size

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less than 40 nm could be prepared using EASAI method. Particle size also can be controlled effectively using PVP or HPMC as polymeric stabilizers. The polymeric stabilizers effectively control the particle size even without strongly interacting with the GF. The reduction of particle size and the presence of the lyophilic polymers led to considerable enhancement in the solubility and drug release rate. The possibility of controlling the particle size, even to the extent of less than 40 nm, without largely compromising the crystallinity makes the EASAI method quite attractive. Acknowledgments Thanks are due to Advanced Materials Research Centre (AMRC), IIT Mandi for providing laboratory facilities. Financial assistance from ARMREB (DRDO), IIT Mandi and UGC (SRF) is gratefully acknowledged.

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