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Effect of Polymer Molecular Weight on the Production of Drug Nanoparticles
Effect of Polymer Molecular Weight on The Production of Drug Nanoparticles S. SEPASSI,1 D.J. GOODWIN,1 A.F. DRAKE,1 S. HOLLAND,2 G. LEONARD,2 L. MARTINI,2 M.J. LAWRENCE1 1
Department of Pharmacy, King’s College London, 150 Stamford Street, London SE1 9NH, United Kingdom
2
GlaxoSmithKline, New Frontiers Science Park, Harlow, Essex CM19 5AW, United Kingdom
Received 4 September 2006; revised 20 December 2006; accepted 3 January 2007 Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.20979
ABSTRACT: Stable, polymer-coated nanoparticles of two hydrophobic drugs, namely nabumetone and halofantrine, have been prepared by a wet-bead milling process performed in the presence of a stabilizing homopolymer, either hydroxypropylmethylcellulose (HPMC) or polyvinylpyrrolidone (PVP), of differing molecular weights and concentrations. Although nabumetone nanoparticles could only be produced when HPMC was used as stabilizing polymer, halofantrine nanoparticles could be prepared using either HPMC or PVP. Stable nanoparticles of nabumetone could be produced using a HPMC solution of viscosity average molecular weight, Mv, of 5 kg/mol over an approximate four fold polymer concentration range (0.63–2.5%w/w) when a drug loading of 20%w/w was used. Increasing the molecular weight of HPMC up to a limiting Mv of 89 kg/mol did not result in the formation of nanoparticles at any of the polymer concentrations examined. The amount of polymer absorbed onto the nanoparticles was determined by measuring the depletion of polymer from solution based on either an ultra-violet (PVP) or optical rotatory dispersion (HPMC) assay. The slightly lower concentration of HMPC found to be present on the surface of the halofantrine nanoparticles compared with the nabumetone nanoparticles suggested a differing affinity of the polymer for the surface of the two drugs. ß 2007 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 96:2655–2666, 2007
Keywords:
colloid; polymers; physical characterization; nanoparticles; formulation
INTRODUCTION Reducing the particle size of a drug to the micron size range has long been acknowledged as a means of increasing the absorption and consequently the bioavailability of poorly water-soluble therapeutic agents including griseofulvin,1 digoxin,2 phenytoin,3 and progesterone.4 Indeed the importance of using a drug particle size sufficiently small enough to obtain as high as possible Correspondence to: M.J. Lawrence (Telephone: þ0207 848 4808; Fax: þ0207 848 4800; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 96, 2655–2666 (2007) ß 2007 Wiley-Liss, Inc. and the American Pharmacists Association
bioavailability is so well recognized that many Pharmacopoeias include a recommendation of an upper particle size limit in cases where poor water-solubility is an issue. Until recently, however, the ability of existing particle size reduction techniques to reduce particle size was limited to sizes within the micron range because of the rapid agglomeration of sub-micron drug particles due to the presence of cohesive forces.5 With the advent of high-pressure homogenization (Dissocube1),6 wet-bead milling (Nanocrystal1),7 and supercritical fluid-based technologies,8 stable drug particles in the sub-micron, or nanometer, size range can now be readily produced, generally in the form of a (nano)suspension which can be
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lyophilized for injectable products or processed as a dry powder for incorporation into solid dosage formulations.9 Utilizing nanoparticles produced using these techniques, a number of in vivo studies have demonstrated the benefit of this large-size reduction for a range of disparate therapeutic compounds that possess a solubilitylimited absorption.10–12 In fact, there are now currently two products on the market based on the Nanocrystal1 technology,13 with many more in development. Here, we report on a wet-bead milling process which achieves particle size reduction to within the nanometer size range by using a high impact bead mill to fracture drug particles dispersed in a crude concentrated suspension. In line with most, if not all, of the techniques used to reduce particle size to sub-micron levels, wet-bead milling requires the presence of a polymer and/ or surfactant to stabilize the resulting nanoparticles against agglomeration. The polymer and/or surfactant adsorbs to the freshly formed drug crystal surfaces during milling, to provide a steric or ionic barrier around the drug nanoparticles. In the present study, the neutral homopolymers hydroxypropylmethylcellulose (HPMC) and polyvinylpyrrolidone (PVP), were used because their stabilizing ability is less dependent than ionic polymers or surfactants, upon changes in the chemical environment, such as changes in pH, as well as the contents that a formulation encounters upon passage down the gastro-intestinal tract.10,11 The beads used in the present milling process were composed of yttrium zirconia (YTZ1) because these beads possess the property of ensuring that contamination of the final product by bead material was at an acceptable level for a pharmaceutical product.14 To our knowledge, there has not been a systematic investigation into the effects of the stabilizing agent on the production of drug nanoparticles by wet-bead milling and why different polymer/ surfactants stabilize different drug nanoparticles. Although adsorption of various types of polymer onto (preformed) nanoparticles has been extensively studied, for example, by Kellaway et al.,15 Tanaka et al.,16 and Cosgrove et al.,17 these studies typically use low volume fractions of well characterized model systems, such as spherical polystyrene or silica particles, and whose surface area remains constant during the adsorption and stabilization process. Extrapolation from these studies to the processes occurring during nanoparticle production using wet-bead milling of
heterogeneous drug particles, which are present at high volume fraction and are constantly changing their size and hence surface area during milling, may not be appropriate. While other studies, such as that of Law and Kayes18 and Duro et al.,19 have looked at the effect of various factors influencing the adsorption and stabilization of drug particles by polymers, the particle size of the drug, which was in the micron size range, again remained constant during the adsorption process again making extrapolation inappropriate. Finally, as a result of their high surface to volume ratio, nanoparticles are considered to possess different physical properties compared with their microparticle counterparts.20 In the present study, two examples of poorly water-soluble drugs that suffer from poor bioavailability, namely nabumetone, a nonsteroidal anti-inflammatory drug and halofantrine, an anti-malarial agent, were selected for an investigation of the factors influencing nanoparticle production. The majority of work focuses on the effect of polymer type and molecular weight of two pharmaceutically acceptable polymers on nanoparticle production.
MATERIALS AND METHODS Materials Nabumetone and halofantrine were supplied by GlaxoSmithKline (Harlow, UK). Nabumetone has an aqueous solubility of 4.7 mg/L at 258C while halofantrine is insoluble in water with a solubility of <1.0 mg/L at 258C (GlaxoSmithKline). HPMC, with a nominal molecular weight of 8 and 86 kg/ mol (Methocel1 E3LV (HPMC 8) and Methocel1 E4M (HPMC 86), respectively), was kindly donated by Colorcon Ltd. (Dartford, UK). HPMC of a nominal molecular weight of 11 and 24 kg/mol (HPMC 11 and HPMC 24, respectively) were purchased from Sigma-Aldrich Co. Ltd. (Poole, UK). PVP, of molecular weights 3.9 kg/mol (Kollidon1 12 (PVP 12)), 42.5 kg/mol (Kollidon1 30 (PVP 30)), and 13,000 kg/mol (Kollidon1 90 (PVP 90)) was supplied by BASF Plc. (Cheshire, UK). Again polymer molecular weights quoted are those claimed by the manufacturer. The manufacturers designated code was used throughout the study. Yttrium zirconia beads of size 0.65 mm (range 0.55–0.69 mm) and 0.44 mm (0.35–0.5 mm range) were obtained from the Nikkato Corp. (Tokyo, Japan). Spectroscopically pure water (18.2 MV) obtained from an Elga Maxima ultra
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pure water unit (High Wycombe, UK) was used for studies of polymer adsorption; distilled water was used in all other experiments. All chemicals were used as received and polymer concentrations were not corrected for any moisture that may be present.
Molecular Weight Characterisation of Polymers An estimate of the viscosity average molecular weight (Mv) of the HPMC and PVP polymers were obtained from capillary viscometry. The amount of polymer used to prepare the stock solutions varied with the molecular weight of the polymer to ensure that measurements were made below C (defined as 1/[h] where [h] is the intrinsic viscosity of the polymer). Polymer solutions were prepared at 5% w/v (PVP 12), 1.0% w/v (PVP 30), 1.2% w/v (HPMC 8), 1.0% w/v (HPMC 11), 0.25% w/v (HPMC 24), 0.1% w/v (HPMC 86) in distilled water. Automated measurements of flow times of the polymer solutions in an Ubbelholde viscometer were carried out using a Viscosity Measuring Unit AVS 350 (Schott-Gera¨te, Hofheim, Germany), connected to a ViscoDoser AVS 20 piston burette (for automatic dilutions). The viscometer was immersed in a precision water bath (transparent thermostat CT 1650, SchottGera¨te, Hofheim, Germany) to maintain the temperature at 20 0.18C. Flow time measurements of each polymer solution were made in triplicate. The intercept of separate Huggins and Kramer extrapolations (linear regression) yielded [h] and from this the molecular weight was determined by applying the Mark-Houwink equation for HPMC with the constants K and a being set to 3.39 104 and 0.88.18 The molecular
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weights (Mv) of the HPMCs used in this study were found to range between 5 and 89 kg/mol (Table 1). For PVP, the K-value (i.e., the Fikentscher K-value, not the constant K of the Mark-Houwink equation) was calculated for each PVP using the equation given in the British Pharmacopeia.21 From the K-value it was possible to calculate the viscosity-average molecular weight (Mv).22 The results of this analysis are given in Table 1. For both types of polymer the results of the viscosity and molecular weight determination are expressed as the mean the standard deviation of three individual measurements. The number (Mn) and weight (Mw) averaged molecular weight of PVP 12, 30, and 90 and of HPMC 8 and HPMC 86 were determined using gel permeation chromatography (GPC) by Rapra Technology (Shrewsbury, UK) (Table 1). For PVP, a solution of the sample was prepared by dissolving 25 mg in 15 cm3 dimethylformamide (DMF). The solution was filtered through a 0.2 mm polyamide membrane into sample vials and placed in an autosampler. GPC was carried out on a Waters 150 CV instrument using a Plgel 2 mixed bed-B, 30 cm, 10 mm column (Polymer Laboratories, Shropshire, UK) using DMF containing 0.01 M lithium bromide as the solvent, at a flow rate of 1.0 cm3/min (nominal) at 808C with a refractive index detector. The data collected was analysed using Viscotek Trisec 3.0 software (Table 1). This method was used in the present study as it has been previously found23 to yield PVP molecular weights that were in good agreement with those obtained by static light scattering measurements. For HPMC, a solution of polymer was made by dissolving in a 0.2 M aqueous
Table 1. Intrinsic Viscosity [h] at (208C) and Viscosity-Average Molecular Weight (Mv) from Capillary Viscosity Measurements (n ¼ 3 s.d.) and Number (Mn) and Weight-Average (Mw) Molecular Weight and Polydispersity ( Pd ¼ Mw/Mn) of Polymer Solutions from GPC (mean, n ¼ 2)
Capillary Viscometry Polymer Identification HPMC 8 HPMC 11 HPMC 24 HPMC 86 PVP 12 PVP 30 PVP 90
GPC
Nominal Molecular Weight (kg/mol)a
[h] (mL/g)
Mv (kg/mol)
Mw (kg/mol)
Mn (kg/mol)
Pd
8 11 24 86 3.9 42.5 1300
61.4 0.2 88.9 1.2 306.2 1.7 770.1 2.9 4.7 0.1 20.8 0.1 N/D
5.0 0.0 7.7 0.1 31.3 0.2 89.1 0.4 3.0 0.1 46.2 0.4 N/D
23.1 45.6 298.0 687.5 3.4 41.0 560
4.2 5.8 44.0 56.2 2.7 16.0 140
5.5 7.9 6.8 12.0 1.2 2.6 4.0
N/D ¼ not determined. a Molecular weight as stated by manufacturer. DOI 10.1002/jps
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solution of ammonium acetate. The resulting solution was filtered through a 0.45 mm PVDF filter directly into sample vials and placed in an autosampler prior to GPC analysis using two Plgel mixed-OH, 30 cm, 8mm columns fitted with a Plgel guard column and a refractive index detector. The GPC analysis was performed at a temperature of 30oC and flow rate of 1.0 cm3/min was employed. A summary of the number (Mn) and weightaverage (Mw) molecular weights determined in this way as well as the polydispersity ( Pd) of the polymers are given in Table 1. The results are expressed as the mean of two separate measurements. In no case did the duplicate measurements deviate by more than 10%.
Production of Nabumetone and Halofantrine Nanoparticles Preparation of Crude Suspensions Polymer solutions were prepared by weighing the appropriate amount of polymer and making to weight with distilled water. Once the polymer had dissolved, sufficient polymer solution was added to either 10 g of nabumetone or 15 g of halofantrine with constant (magnetic) stirring to form 50 g of crude suspension or slurry. It should be noted that the polymer concentrations are generally quoted in the text as the weight % of polymer in the solution, which was added to drug to form a crude suspension prior to milling and not the final concentration in the crude drug suspension.
nabumetone suspension with 1.88% w/w HPMC 8 (NB the wet-bead milling process requires a high drug loading). Varying volumes of beads and crude suspension were added to a 25 cm3 volume milling jar made from food-grade nylon (Nylacast, Leicester, UK) and milled for 6 h using a Retsch MM200 Mixer Mill (Glen Creston, Stanmore, UK) at 30 vibrations/s. For each condition studied three separate suspensions were prepared and milled and, therefore, the results are expressed as the mean of three measurements the standard deviation (Table 2). ‘‘Optimum’’ process parameters of 10 cm3 of 0.44 mm beads and 10 cm3 of crude drug suspension were selected on the basis of being the protocol which yielded the minimum nanoparticle size obtained as assessed by laser diffraction after 6 h milling; milling for longer periods did not appear to cause any further significant reduction in particle size. These parameters were used for the milling of all subsequent formulations.
Physical Characterization of Nanoparticles Particle Size Analysis: Laser Diffraction
Wet-Bead Milling
Particle size analysis of nanoparticles was performed using a Malvern 2600 series laser diffractometer (Malvern Instruments, Malvern, UK). Nanosuspensions were diluted in distilled water to obtain an acceptable obscuration level for size determination. From the particle size data, the volume (or mass) moment mean (D[4,3]) was used to characterize the nanoparticles (Eq. 1). P 4 d D½4; 3 ¼ P 3 (1) d
A preliminary study into the effect of bead loading, slurry (crude suspension) loading, and bead size was undertaken using a 20% w/w
The volume moment mean was chosen for this study as it is sensitive to the presence of large
Table 2. Effect of Slurry and Bead Loading and Bead Size on the Mean Particle Size of nabumetone after 6 h of wet-bead milling in the presence of 1.88% w/w HPMC 8 (n ¼ 3 s.d.)
Bead Loading (mL)
Slurry Loading (mL)
Bead Size (mm)
Mean Particle Sizea (mm)
12 8 7 10 12 8 7 10
0.44 0.44 0.44 0.44 0.65 0.65 0.65 0.65
0.79 0.04 0.74 0.00 0.69 0.06 0.65 0.06 0.79 0.04 0.75 0.01 0.77 0.03 0.76 0.01
7 7 12 10 7 7 12 10 a
Obtained from laser diffraction.
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particles in the sample24 and allows us to establish whether any large particles are still present in the nanosuspension because even if they are small in terms of their number, they would make up a considerable mass of the drug particles. (It should be mentioned that, because of the polydispersity of the nanoparticles and the assumption of spherical particles, the mean size of the particles determined depends upon the precise technique used to measure particle size, for example, use of photon correlation spectroscopy (Brookhaven ZetaPlus Sizer 2.29) yielded nanoparticle sizes about half that obtained using laser diffraction). Electron Microscopy A drop of milled nanosuspension was placed on the surface of a small (ca. 5 5 mm) piece of freshly cleaved mica and allowed to dry. This was then mounted onto a specimen stub using double-sided adhesive tape, and coated with a layer of gold approximately 15 nm thick using a Polaron E5100 sputter coater. A Philips SEM501B scanning electron microscope operating at 15 kV was used to visualize the samples. Freeze fracture microscopy was carried out on nanosuspensions of 20% w/w nabumetone prepared using a 1.25% w/w solution of HPMC 8, 30% w/w halofantrine with 1.43% w/w HPMC 8 and 30% w/w halofantrine prepared with 1.43% w/w PVP30. Suspensions were prepared according to the method described above. Subsequent to 6 h wet-bead milling, the nanoparticle suspensions were subject to size analysis and then centrifuged at 13,000 rpm for 1.5 h. Determination of Polymer Concentration A series of solutions were prepared containing 0.01, 0.1, 1, 3, and 5% w/w of either HPMC 8, HPMC 11, or HPMC 24 in ultra-pure water. The UV spectra of the solutions were obtained over a wavelength range of 180–600 nm on a p180 spectrophotometer (Applied Photophysics Ltd., Leatherhead, UK.). The instrument was continually flushed with nitrogen. All samples, including ultra-pure water used in the preparation of standard samples (prepared and measured in triplicate) were scanned against air and the absorbance of water subtracted from that of the HPMC containing samples in order to obtain the UV spectra of HPMC. Optical rotatory dispersion (ORD) spectra were obtained using a Jasco J600 spectropolarimeter (Jasco UK Ltd., DOI 10.1002/jps
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Great Dunmow, UK) with a spectral bandwidth of 2 nm, scanning rate of 20 nm/min and data resolution of 1 nm. Solutions to be measured were placed in a 5 cm cell and the ORD measured over a wavelength range of 650–230 nm. The values of the ORD at 350 nm were plotted as a function of HPMC concentration. The assay for PVP was based on UV spectroscopy. A range of solutions of different PVPs namely PVP 12, 30, and 90 were prepared from a stock solution in spectroscopically pure water. Each dilution was prepared at least in triplicate. The solutions were scanned in a cuvette with 0.1 cm pathlength over a wavelength range of 185–400 nm using a p180 spectrophotometer with a spectral bandwidth of 2 nm, at a scanning rate of 50 nm/min and a step size of 1 nm. All samples, including the water used to prepare the samples were scanned against air and the absorbance of water subtracted from that of the PVP containing samples. The UV absorbance at a wavelength of 195 nm was plotted as a function of concentration. Determination of Polymer Adsorption The amount of HPMC 8 adsorbed onto nabumetone nanoparticles was obtained by measuring its depletion from solution using the following procedure. Ten cubic centimeter of aqueous suspensions comprising 20% w/w nabumetone and various concentrations of HPMC 8 were milled for 6 h using a Restch MM200 mill with 10 cm3 of 0.44 mm beads. The resulting nanosuspensions were then centrifuged at 13,000 rpm for 1.5 h. The supernatants were collected and assayed for HPMC content by measuring the ORD spectrum over 650–230 nm using a 2 cm path length cell. A smaller path length cell than was used for calibration purposes was necessitated for examining the test sample due to the low volume of polymer solution collected after removal of the drug and adsorbed polymer. Furthermore, due to the low volume of nanosupsensions produced after milling, the ORD of each test sample could only be measured once. Therefore, three individual nanosupensions were prepared and the ORD of each measured so that the concentration of HPMC 8 remaining in solution after adsorption could be expressed as the mean s.d. of three determinations. By determination of the concentration of polymer remaining in the supernatant, it is possible to calculate the amount of polymer lost from solution
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and thereby determine the amount of polymer adsorbed onto the drug. In order to determine the amount of polymer adsorbed an estimate of the total surface area of the drug nanoparticles was made based on particle size analysis by laser diffraction, assuming the drug particles to be spherical, and using the density of the drug determined using a StereopycnometerTM (Quantachrome Instruments, Hook, UK). The pycnometer uses Archimede’s principle of fluid (gas) displacement and the technique of gas expansion. In the present study, helium was used as the displacing fluid (gas). The density of nabumetone was determined to be 1.19 g/cm3 and halofantrine to be 1.39 g/cm3. The same methodology was repeated to obtain adsorption isotherms for halofantrine with HPMC 8 or PVP 30. However, the amount of PVP left in solution after the adsorption was complete was quantified using UV spectroscopy as described above.
RESULTS AND DISCUSSION Effect of Polymer and Concentration of Nanoparticle Production Size analysis of nabumetone particles after 6 h milling in the presence of HMPC shown in Table 3. Note that nabumetone nanoparticles could not be prepared if PVP was used as stabling polymer. Also given in Table 3 is the weight ratio of drug:polymer used in the present study. This
value is reported in the present study because it has been stated by other workers, for example, Merisko-Liversidge et al.9 and Date and Patravale,25 as being an important factor when preparing nanoparticles. These workers quote, respectively, that a drug:stabilizer weight ratio in the range of 20:1 to 2:1 or a maximum ratio of drug:stabilizer of 1.7:1 as being necessary for nanoparticle production. Although the weight ratio of drug:stabilizer used for nanoparticle production might be a useful parameter for the formulator, the present authors do not find it particularly helpful in understanding the mechanism of polymer adsorption since it neglects amongst other things the density of the drug, the size of the nanoparticles, and the molecular weight of the polymer. Furthermore, as can be seen from Table 3 there are a number of instances in the present study where nanoparticles were produced using weight ratios of drug:polymer outside the quoted range. From Table 3, it is clear that for each polymer molecular weight that a minimum and maximum concentration of HPMC is required to produce and stabilize the nabumetone nanoparticles. At HPMC concentrations below the minimum required for production of nabumetone nanoparticles, it is probable that there is insufficient polymer present to completely cover the surface of the drug particle in order to provide a steric barrier against the cohesive forces between the drug particles that lead to the aggregation and agglomeration of these small sized particles. At the other end of the concentration range, at
Table 3. Effect of Polymer Molecular Weight on the Mean Particle Size of Nabumetone Milled for 6 h in the Presence of Various Concentrations of HPMC (n ¼ 3 s.d.)
Nominal HPMC Molecular Weight (g/mol)/Mean Particle Size of Nabumetone after 6 h Bead Milling (mm) HPMC Concentration (% w/w) 0.06 0.13 0.63 0.94 1.25 1.56 1.88 2.50 3.75 6.25
Weight Ratio of Drug to Polymer
HPMC 8
HPMC 11
HPMC 24
HPMC 86
417:1 192:1 40:1 27:1 20:1 16:1 13:1 10:1 7:1 4:1
17.80 1.03 2.96 0.07 0.75 0.01 0.86 0.02 0.65 0.04 0.87 0.01 0.65 0.4 0.90 0.01 1.27 0.29 1.81 0.88
ND 6.93 3.52 0.89 0.03 0.74 0.01 0.86 0.01 0.96 0.03 0.78 0.01 1.16 0.04 2.42 0.07 ND
ND 3.32 0.12 2.19 0.12 2.19 0.03 0.94 0.01 1.61 0.04 1.98 0.19 ND ND ND
18.69 9.04 11.51 1.39 2.67 0.15 2.31 0.04 Solid Solid Solid Solid Solid Solid
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stabilizer levels above the maximum HPMC concentration that produces nabumetone nanoparticles, the average size of the drug particles produced by milling increased to above 1 mm and, therefore, the anticipated benefits in dissolution rate of the nanoparticles lost. As can be seen from Table 3, the concentration range over which HPMC could produce nanoparticles was found to decrease with an increase in the molecular weight of the polymer. For example, using HPMC 24, nabumetone nanoparticles could only be produced at a concentration of 1.25% w/w whereas it was not possible to produce nanoparticles at any of the concentrations studied when using HPMC 86 as the stabilizing polymer. The reduction observed in the maximum concentration of polymer that can be used to produce nanoparticles as the molecular weight of the stabilizing polymer is increased is thought to be, at least in part, a consequence of the accompanying increase in the viscosity of the suspension. It has been suggested that since the milling process relies upon the impaction of beads on drug particles increasing the viscosity of the suspension being milled may lead to the beads traveling at a lower velocity, thereby resulting in smaller forces of impaction and, therefore, less efficient size reduction.26,27 This hypothesis is in agreement with simulations of the motion of balls in a vibration mill performed by Yokoyama et al.28 in which an increase in the viscosity of the surrounding medium caused a radical drop in the intensity of ball collisions. The observation that the minimum concentration of HPMC required for nanoparticle production also increases with the higher molecular weight grades can be partly explained by a combination of the slow diffusion of the higher molecular weight polymers to the ‘‘bare’’ drug surfaces created during milling and the occurrence of bridging flocculation at these relatively low polymer concentrations. Bridging flocculation is where a single polymer molecule is adsorbed to more than one particle.29 The inability of the higher molecular weight polymers to prepare nanoparticles may have important implications for the stability of the resulting nanoparticles as it is well established that higher molecular weight polymeric stabilizers tend to be favored since they are reported to yield a more stable dispersion compared to lower molecular weight grades of the same polymer. This is generally attributed to the fact that higher molecular weight polymers form a thicker adsorbed layer and thus offer greater DOI 10.1002/jps
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stabilization upon approaching particles than lower molecular weight polymers.30 By way of comparison between the two drugs studied, when halofantrine was milled in the presence of HPMC 8 and HPMC 24; nanoparticles could be formed between 0.71% w/w and 2.14% w/ w HMPC 8 (HPMC 8 concentrations outside this range were not examined) and at 0.14% w/w HPMC 24 (surprisingly nanoparticles were not produced at HPMC 24 concentrations of 0.71 and 1.43% w/w). Unfortunately, due to a limited supply of halofantrine it was not possible to perform the experiments needed to allow the upper and lower limit of stabilizing polymer concentration to be obtained. Significantly, however, it was not possible to prepare nabumetone nanoparticles using only 0.14% w/w HPMC 24, which suggests a different mechanism of coating of the two types of nanoparticles by HPMC, especially bearing in mind the higher drug loading/lower amount of polymer present in the halofantrine preparation. In contrast to nabumetone (data not shown), it was also found possible to produce halofantrine nanoparticles using either PVP 12 or PVP 30 as a stabilizer in the range 0.71–2.14% w/w, although the upper and lower concentration limits were not found due to constraints on the amount of halofantrine available. It is worth commenting that the nanoparticles prepared using the present methodology were stable with respect to size when stored at 258C for at least 1 year, longer periods were not studied (data not shown). No evidence of Ostwald ripening was seen in any of the nanosuspensions tested. This observation agrees with that of MeriskoLiversidge et al.9 who reported that in the liquid state such polymer stabilized nanoparticles were very stable, especially if the solubility of the drug was less than 1 mg/cm3 (as in the present study). Furthermore, the nanoparticles were stable regardless of the concentration at which the nanoparticles were stored, that is, whether they were stored at the concentration at which they were prepared or stored in a diluted form. In addition, the nanoparticles could be readily resuspended after centrifugation and replacement of the excess polymer solution with an equal volume of water; the nanoparticles in this state were also stable for periods of at least 1 year. All nanosuspensions, however, did exhibit sedimentation upon standing, although as expected, the sedimentation rate in the nanosuspensions containing excess polymer was slower than in its absence. In addition, HPMC stabilized
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nanoparticles remained uniformly dispersed for longer than the PVP-stabilized ones, which tended to form a sediment which was harder to redisperse. Morphology of Nanoparticles The SEM images of nabumetone nanoparticles milled for 6 h in the presence of 1.25% w/w HPMC 8 (Fig. 1) and 1.25% w/w HPMC 24 (Fig. 2). As can be seen in Figures 1 and 2 the majority of nanoparticles were polydisperse rods. Reassuringly an estimate of the size of the nabumetone nanoparticles based on the SEM images confirms the general findings from laser diffraction size analysis, with the nanoparticles prepared using HPMC 24 exhibiting the slightly larger size. It is worth commenting that the somewhat melted appearance of the nabumetone nanoparticles is thought to be a consequence of the nanoparticles melting in the vacuum of the electron microscope (the melting point of nabumetone at 1 atmosphere is 78–838C). Figure 3 shows the SEMs obtained of halofantrine nanoparticles milled in the presence of either 1.43% w/w HPMC 8 or 1.43% w/w PVP 30. As can be seen the halofantrine nanoparticles milled in the presence of HPMC 8 appeared more irregular in shape than the rod-shaped nabumetone nanoparticles prepared using HPMC 8 (Fig. 1). In addition, they varied from the halofantrine nanoparticles formed in the presence of PVP 30, which appeared cuboidal at larger sizes, and spherical at smaller sizes, possibly due to the
Figure 2. SEM of nabumetone suspension milled in the presence of 1.25% w/w HPMC 24 for 6 h.
attrition of the hard edges of the particle as milling progresses. According to Merisko-Liversidge et al.,9 the geometrical shape of milled drug nanoparticles is dependent upon the morphology of the starting drug material, the fracture plane of the crystals and drug/stabilizer interactions. Although the influence of the original shape of the drug crystal in determining the final shape of the nanoparticles has not been examined in the present study, it does appear that the type of stabilizer used does have an affect on the shape of the resulting nanoparticles based on halofantrine nanoparticle SEM images. Adsorption Isotherms
Figure 1. SEMs of nabumetone suspension milled in the presence of 1.25% w/w HPMC 8 for 6 h.
Quantification of the amount of polymer adsorbed onto the drug nanoparticles was made using the depletion method. This method involves determining the concentration difference of polymer in the continuous phase before and after milling and relating this to the surface area of drug available for adsorption. The available surface area of the drug is estimated by determining the number and size of the nanoparticles from the laser diffraction studies assuming spherical nanoparticles. Unfortunately, in the present study, the nonspherical polydisperse nature of the nanoparticles (as seen in the SEMs of both nabumetone and halofantrine nanoparticles, Figs. 1–3) will introduce a degree of error into this calculation. In order to estimate how much polymer has adsorbed onto the drug, a suitable analytical technique is required for the adsorbate.31
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Figure 3. SEM images of halofantrine nanoparticles comparing the effect of the stabiliser on nanoparticle morphology (a) halofantrine nanoparticles prepared with 1.43% w/w HPMC 8 and (b) halofantrine nanoparticles prepared with 1.43% w/w PVP 30.
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Although colourimetric assays are available for both PVP32 and HPMC (USP23 assay of Hypromellose Ophthalmic Solution),33 such assays are very dependent upon the molecular weight of the polymer being assayed, particularly when using relatively low molecular weight polymers such as those used in the present study. The nonmolecular weight dependence of the assay is essential in the present study because fractionation of the polymer is reported to occur at the solid–liquid interface due to the preferential adsorption of longer polymer chains.34 In the light of these findings, an ORD assay was developed for HPMC and UV spectroscopy was used to quantify the concentration of PVP in solution. The UV assay developed was independent of polymer molecular weight over the nominal weight range of 3.9– 1,300 kg/mol (data not shown). A critical requirement for nanoparticle production is that the stabilizing agent adsorbs to the surface of the drug particle to prevent agglomeration.7 Establishment of the adsorbed amount is, therefore, an important parameter to characterize polymer-stabilized nanoparticles, the extent of adsorption being based on competing interactions between the drug particle surface, the polymer, and the solvent molecules.35 The dependence of the amount of polymer adsorbed onto the drug after 6 h of milling on the initial concentration of HPMC 8 is given in Table 4. As can be seen from Table 4, the profile of the adsorption isotherm is different to those commonly reported in the literature31 since the initial (rising) portion of the isotherm is absent due to the fact that nanoparticles can only be produced once the concentration of stabilizer is above a certain threshold in order to completely coat the surface of the drug particle, discussed above. At a starting concentration of 0.63% w/w HPMC 8, it can be seen from Table 4 that a polymer concentration of 0.17% w/w was left in solution, so that as a consequence 76 mg of HPMC 8 was adsorbed on to 4 g of
Table 4. Mean Adsorption of HPMC 8 on Milled Nabumetone Particles at Various Concentrations (n ¼ 3 s.d.)
Starting Polymer Concentration (%w/w) 0.63 0.94 1.25 1.56 1.88 DOI 10.1002/jps
Mean Concentration of Polymer in Solution after Adsorption (%w/w)
Mean Amount of Polymer Adsorbed onto Drug (mg)
Mean Amount of Polymer Adsorbed onto Drug (mg/m2)
0.17 0.08 0.31 0.15 0.55 0.25 1.20 0.25 1.16 0.10
76.0 14.0 105.4 24.7 115.9 8.4 59.9 42.0 119.2 17.0
2.8 0.5 4.5 1.1 3.7 0.3 2.6 1.8 3.8 0.6
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Table 5. Mean Adsorption of HPMC 8 on Milled Halofantrine Particles at Various Concentrations (n ¼ 3 s.d.)
Starting Polymer Concentration (%w/w) 0.71 1.07 1.43 1.79 2.14
Mean Concentration of Polymer in Solution after Adsorption (%w/w)
Mean Amount of Polymer Adsorbed onto Drug (mg)
Mean Amount of Polymer Adsorbed onto Drug (mg/m2)
0.09 0.05 0.35 0.02 0.81 0.02 1.08 0.23 1.44 0.19
97.8 7.9 112.9 2.7 96.7 3.6 111.4 35.3 109.8 29.1
2.5 0.2 2.8 0.1 2.7 0.1 2.7 0.9 3.1 0.8
nabumetone nanoparticles. This value represents the minimum amount of polymer required to stabilize nabumetone nanoparticles. Particle size analysis results (Table 3) confirm this finding since milling of nabumetone with a concentration of HPMC less than 0.46% w/w, for example, 0.13% w/w, led to the production of micro- rather than nanoparticles. By making the assumptions described above, it is possible to determine the mg of drug absorbed per m2 of drug. It can be seen from Table 4 that increasing the starting concentration of HPMC had little effect on the amount of polymer adsorbed, suggesting that the absorption plateau had been reached. It is worth noting that the adsorbed amount of polymer is much larger than is normally quoted in the literature for comparable systems, e.g. polymer adsorption on polystyrene nanoparticles. This discrepancy is undoubtedly partly (not but not solely) due to the assumption of monodisperse spherical particles, which will underestimate the surface area of drug available for absorption. The adsorption of HPMC 8 on halofantrine (Table 5) resulted in a slightly lower plateau value than HPMC 8 on nabumetone and may be attributed to a difference in affinity of HPMC 8 for the surface of the two drugs. A lower adsorption of PVP 30 was found on halofantrine (Table 6) compared with HPMC 8 suggesting that smaller amounts of PVP 30 than HPMC 8 are required to stabilize the halofantrine nanoparticles. As both
HPMC and PVP are nonionic polymers and the two drugs studied are neutral species, the forces of attachment of these polymers onto the surface of the drugs can be attributed to either hydrophobic interactions or via hydrogen bonding.36 In order to ensure that the assumptions used in estimating adsorbed amount did not alter the trends of the results, calculations were also performed assuming the presence of nanoparticles of equivalent volume to the spheres measured using laser diffraction but approximating the shape of nanoparticles to that seen using FFEM, namely cubes for halofantrine nanoparticles prepared in the presence of PVP 30 and rodlike particles (length to width ratio of 3:1) for both nabumetone and halofantrine nanoparticles milled with HPMC 8. As the milled nanoparticles are not regular in shape this approach is also in error but serves to illustrate that the differences observed between the different combinations of drug and polymer are genuine and not due to the assumptions of nanoparticle shape made in the present study. As would be anticipated (from surface area:volume considerations), the amount absorbed was greatest assuming spherical particles (minimum surface area to volume) was 20% less when cuboids were assumed and 28% less when rodlike particles were assumed. While the amount adsorbed obviously varied depending upon the shape of the nanoparticles assumed, the trend of the results obtained assuming
Table 6. Mean Adsorption of PVP 30 on Milled Halofantrine Particles at Various Concentrations (n ¼ 3 s.d.)
Starting Polymer Concentration (%w/w) 0.71 1.07 1.43 1.79 2.14
Mean Concentration of Polymer in Solution after Adsorption (%w/w)
Mean Amount of Polymer Adsorbed onto Drug (g)
Mean Amount of Polymer Adsorbed onto Drug (mg/m2)
0.46 0.02 0.75 0.06 1.18 0.07 1.48 0.05 1.92 0.08
39.2 3.1 50.2 9.4 39.2 11.0 48.6 7.8 34.5 12.6
1.0 0.1 1.4 0.3 1.2 0.3 1.4 0.2 0.9 0.3
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spherical nanoparticles described below does not: with cuboidal halofantrine nanoparticles milled in the presence of PVP 30 exhibiting about a thirdhalf of the absorbed amount of rodlike nabumetone and halofantrine nanoparticles milled in the presence of HPMC 8. The differences in adsorbed amount recorded for the different drug/polymer combinations may reflect differences in the conformation of the adsorbed polymer on the surface of the drug nanoparticles. These differences may be due to the differing nature of the two polymers and/or differences in polymer molecular weight (Table 1). Further work such as small angle neutron scattering studies is required to determine the conformation of the polymer on the surface of the drug nanoparticles.
CONCLUSION Higher molecular weight polymers tend to be favored for steric stabilization since they are reported to yield a more stable dispersion compared to lower molecular weight grades of the same polymer. As the method of producing nanoparticles using wet-bead milling demands a high drug loading, this precludes the use of high molecular weight polymeric stabilizers due to the resulting high viscosity of the milling slurry. However, in the present study it has been found that the molecular weight of the polymer used did not influence drug nanoparticle stability.
ACKNOWLEDGMENTS The authors would like to acknowledge the help of Professor SM Ross-Murphy and Dr. D Picout in performing the viscosity measurements, and Dr. T Bui in developing the analytical technique for assay of PVP and HPMC. The authors also express their appreciation to Dr. A Brain at the KCL Electron Microscopy Unit for his assistance in obtaining the SEM and FFEMs. In addition, SS and DG thank EPSRC, Impact Faraday and GSK for the award of studentships.
REFERENCES 1. Atkinson RM, Bedford C, Child KJ, Tomich EG. 1962. Effect of particle size on blood griseofulvin levels in man. Nature 193:588–589.
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2. Shaw TRD, Carless JE, Howard MR, Raymond K. 1973. Particle size and absorption of digoxin. Lancet 2:209–210. 3. Yakou S, Yamazaki S, Sonobe T, Sugihara M, Fukumuro K, Nagai T. 1986. Particle-size dependency of dissolution rate and human bioavailability of phenytoin in powders and phenytoin polyethylene-glycol solid dispersions. Chem Pharm Bull 34:4400– 4402. 4. Hargrove JT, Maxson WS, Wentz AC. 1989. Absorption of oral progesterone is influenced by vehicle and particle-size. Am J Obstet Gynecol 161:948– 951. 5. Chaumeil JC. 1998. Micronization: A method of improving the bioavailability of poorly soluble drugs. Methods Find Exp Clin Pharmacol 20:211– 215. 6. Mu¨ller RH, Becker R, Kruss B, Peters K. 1998. Pharmaceutical nanosuspension for medicament administration as system of increased saturation solubility and rate of solution. US Patent No. 5858410. 7. Liversidge GG, Cundy KC, Bishop JF, Czekai DA. 1992. Surface modified drug nanoparticles. US Patent No. 5145684. 8. Vasukumar K, Bansal A. 2003. Supercritical fluid technology in pharmaceutical research. CRIPS 4:8– 12. 9. Merisko-Liversidge E, Liversidge GG, Cooper ER. 2003. Nanosizing: A formulation approach for poorly-water soluble compounds. Eur J Pharm Sci 18:113–120. 10. Liversidge GG, Conzentino P. 1995. Drug particle size reduction for decreasing irritancy and enhancing absorption of naproxen in rats. Int J Pharm 125:309–313. 11. Liversidge GG, Cundy KC. 1995. Particle size improvement of oral bioavailability of hydrophobic drugs I. Absolute oral bioavailability of nanocrystalline danazol in beagle dogs. Int J Pharm 125: 91–97. 12. Merisko-Liversidge E, McGurk SL, Liversidge GG. 2004. Insulin nanoparticles: A novel formulation approach for poorly water soluble Zn-insulin. Pharm Res 21:1545–1553. 13. Kipp JE. 2004. The role of solid nanoparticle technology in the parenteral delivery of poorly watersoluble drugs. Int J Pharm 284:109–122. 14. Ruddy SB, Roberts ME. 1998. Nanoparticles containing the R(S) enantiomer of ibuprofen. US Patent No. 5718919. 15. Kellaway IW, Najib NM. 1980. The adsorption of hydrophilic polymers at the polystyrene-water interface. Int J Pharm 6:285–294. 16. Tanaka R, Williams PA, Meadows J, Phillips GO. 1992. The adsorption of hydroxyethyl cellulose and hydrophobically modified hydroxyethyl cellulose onto polystyrene latex. Coll Surf 66:63–72.
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17. Cosgrove T, Mears SJ, Obey T, Thompson L, Wesley RD. 1999. Polymer, particle, surfactant interactions. Coll Surf A 149:329–338. 18. Law SL, Kayes JB. 1983. Adsorption of non-ionic water-soluble cellulose polymers at the solid-water interface and their effect on suspension stability. Int J Pharm 15:251–260. 19. Duro R, Alvarez C, Martinez-Pacheco R, GomezAmoza JL, Concheiro A, Souto C. 1998. The adsorption of cellulose ethers in aqueous suspensions of pyrantel pamoate: Effects on zeta potential and stability. Eur J Pharm Biopharm 45:181– 188. 20. Lee J. 2003. Drug nano- and microparticles processes into solid dosage forms: Physical properties. J Pharm Sci 92:2057–2068. 21. British Pharmacopoeia2005. London: Her Majesty’s Stationary Office. 22. Bu¨ler V, Klodwig U. 1984. Characterizing the molecular weight of polyvinylpyrrolidone. Acta Pharm Tech 30:317–324. 23. Sultana S. 2000. Comparision between polyvinylpyrrolidone-based and polyoxazoline nonionic surfactants: Their physico-chemical and solubilisation behaviour PhD Thesis London University. 24. Rawle A. 2002. The advances in particle sizing to the coatings industry part 1: Particle size measurement. Adv Col Sci Tech 5:1–12. 25. Date AD, Patravale VB. 2004. Current strategies for engineering drug nanoparticles. Curr Opin Coll Int Sci 9:222–235.
26. Parrott EL. 1974. Milling of Pharmaceutical Solids. J Pharm Sci 63:813–829. 27. Denison B. 1990. Bead milling—A practical guide. J Oil Col Chem Assoc 6:256–261. 28. Yokoyama T, Tamura H, Usui H, Jimbo G. 1996. Simulation of ball behaviour in a vibration mill in relation with its grinding rate: Effects of fractional ball filling and liquid viscosity. Int J Miner Process 44–45:413–424. 29. Lochhead RY. 1992. Water soluble polymers solution adsorption and interaction characteristics. Cosmet Toiletries 107:131–156. 30. Sato T. 1993. Stability of dispersion. J Coating Technol 65:113–120. 31. Fleer GJ, Cohen Stuart MA, Scheutjens JMHM, Cosgrove T, Vincent B. 1993. Polymers at Interfaces. London: Chapman and Hall. 32. Bu¨ler V. 1996. Kollidon: Polyvinylpyrrolidone for the Pharmaceutical Industry Ludwigshafen: BASF. 33. United States Pharmacopoeia 231995. US Pharmacopoeial Convention, Rockville, MD. 34. Radeva T. 2002. Adsorption of polymer at colloid surfaces. In: Hubbard A, editor. Encyclopedia of Surface and Colloid Science. New York: Marcel Dekker. p 547–557. 35. Sawyer CB, Reed JS. 2001. Adsorption of hydroxypropyl methyl cellulose in an aqueous system containing multicomponent oxide particles. J Am Ceram Soc 84:1241–1249. 36. Molyneux P. 1983. Water-Soluble Synthetic Polymers: Properties and Behaviour. Volume II. Florida: CRC Press.
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Department of Pharmacy, King’s College London, 150 Stamford Street, London SE1 9NH, United Kingdom
2
GlaxoSmithKline, New Frontiers Science Park, Harlow, Essex CM19 5AW, United Kingdom
Received 4 September 2006; revised 20 December 2006; accepted 3 January 2007 Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.20979
ABSTRACT: Stable, polymer-coated nanoparticles of two hydrophobic drugs, namely nabumetone and halofantrine, have been prepared by a wet-bead milling process performed in the presence of a stabilizing homopolymer, either hydroxypropylmethylcellulose (HPMC) or polyvinylpyrrolidone (PVP), of differing molecular weights and concentrations. Although nabumetone nanoparticles could only be produced when HPMC was used as stabilizing polymer, halofantrine nanoparticles could be prepared using either HPMC or PVP. Stable nanoparticles of nabumetone could be produced using a HPMC solution of viscosity average molecular weight, Mv, of 5 kg/mol over an approximate four fold polymer concentration range (0.63–2.5%w/w) when a drug loading of 20%w/w was used. Increasing the molecular weight of HPMC up to a limiting Mv of 89 kg/mol did not result in the formation of nanoparticles at any of the polymer concentrations examined. The amount of polymer absorbed onto the nanoparticles was determined by measuring the depletion of polymer from solution based on either an ultra-violet (PVP) or optical rotatory dispersion (HPMC) assay. The slightly lower concentration of HMPC found to be present on the surface of the halofantrine nanoparticles compared with the nabumetone nanoparticles suggested a differing affinity of the polymer for the surface of the two drugs. ß 2007 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 96:2655–2666, 2007
Keywords:
colloid; polymers; physical characterization; nanoparticles; formulation
INTRODUCTION Reducing the particle size of a drug to the micron size range has long been acknowledged as a means of increasing the absorption and consequently the bioavailability of poorly water-soluble therapeutic agents including griseofulvin,1 digoxin,2 phenytoin,3 and progesterone.4 Indeed the importance of using a drug particle size sufficiently small enough to obtain as high as possible Correspondence to: M.J. Lawrence (Telephone: þ0207 848 4808; Fax: þ0207 848 4800; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 96, 2655–2666 (2007) ß 2007 Wiley-Liss, Inc. and the American Pharmacists Association
bioavailability is so well recognized that many Pharmacopoeias include a recommendation of an upper particle size limit in cases where poor water-solubility is an issue. Until recently, however, the ability of existing particle size reduction techniques to reduce particle size was limited to sizes within the micron range because of the rapid agglomeration of sub-micron drug particles due to the presence of cohesive forces.5 With the advent of high-pressure homogenization (Dissocube1),6 wet-bead milling (Nanocrystal1),7 and supercritical fluid-based technologies,8 stable drug particles in the sub-micron, or nanometer, size range can now be readily produced, generally in the form of a (nano)suspension which can be
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lyophilized for injectable products or processed as a dry powder for incorporation into solid dosage formulations.9 Utilizing nanoparticles produced using these techniques, a number of in vivo studies have demonstrated the benefit of this large-size reduction for a range of disparate therapeutic compounds that possess a solubilitylimited absorption.10–12 In fact, there are now currently two products on the market based on the Nanocrystal1 technology,13 with many more in development. Here, we report on a wet-bead milling process which achieves particle size reduction to within the nanometer size range by using a high impact bead mill to fracture drug particles dispersed in a crude concentrated suspension. In line with most, if not all, of the techniques used to reduce particle size to sub-micron levels, wet-bead milling requires the presence of a polymer and/ or surfactant to stabilize the resulting nanoparticles against agglomeration. The polymer and/or surfactant adsorbs to the freshly formed drug crystal surfaces during milling, to provide a steric or ionic barrier around the drug nanoparticles. In the present study, the neutral homopolymers hydroxypropylmethylcellulose (HPMC) and polyvinylpyrrolidone (PVP), were used because their stabilizing ability is less dependent than ionic polymers or surfactants, upon changes in the chemical environment, such as changes in pH, as well as the contents that a formulation encounters upon passage down the gastro-intestinal tract.10,11 The beads used in the present milling process were composed of yttrium zirconia (YTZ1) because these beads possess the property of ensuring that contamination of the final product by bead material was at an acceptable level for a pharmaceutical product.14 To our knowledge, there has not been a systematic investigation into the effects of the stabilizing agent on the production of drug nanoparticles by wet-bead milling and why different polymer/ surfactants stabilize different drug nanoparticles. Although adsorption of various types of polymer onto (preformed) nanoparticles has been extensively studied, for example, by Kellaway et al.,15 Tanaka et al.,16 and Cosgrove et al.,17 these studies typically use low volume fractions of well characterized model systems, such as spherical polystyrene or silica particles, and whose surface area remains constant during the adsorption and stabilization process. Extrapolation from these studies to the processes occurring during nanoparticle production using wet-bead milling of
heterogeneous drug particles, which are present at high volume fraction and are constantly changing their size and hence surface area during milling, may not be appropriate. While other studies, such as that of Law and Kayes18 and Duro et al.,19 have looked at the effect of various factors influencing the adsorption and stabilization of drug particles by polymers, the particle size of the drug, which was in the micron size range, again remained constant during the adsorption process again making extrapolation inappropriate. Finally, as a result of their high surface to volume ratio, nanoparticles are considered to possess different physical properties compared with their microparticle counterparts.20 In the present study, two examples of poorly water-soluble drugs that suffer from poor bioavailability, namely nabumetone, a nonsteroidal anti-inflammatory drug and halofantrine, an anti-malarial agent, were selected for an investigation of the factors influencing nanoparticle production. The majority of work focuses on the effect of polymer type and molecular weight of two pharmaceutically acceptable polymers on nanoparticle production.
MATERIALS AND METHODS Materials Nabumetone and halofantrine were supplied by GlaxoSmithKline (Harlow, UK). Nabumetone has an aqueous solubility of 4.7 mg/L at 258C while halofantrine is insoluble in water with a solubility of <1.0 mg/L at 258C (GlaxoSmithKline). HPMC, with a nominal molecular weight of 8 and 86 kg/ mol (Methocel1 E3LV (HPMC 8) and Methocel1 E4M (HPMC 86), respectively), was kindly donated by Colorcon Ltd. (Dartford, UK). HPMC of a nominal molecular weight of 11 and 24 kg/mol (HPMC 11 and HPMC 24, respectively) were purchased from Sigma-Aldrich Co. Ltd. (Poole, UK). PVP, of molecular weights 3.9 kg/mol (Kollidon1 12 (PVP 12)), 42.5 kg/mol (Kollidon1 30 (PVP 30)), and 13,000 kg/mol (Kollidon1 90 (PVP 90)) was supplied by BASF Plc. (Cheshire, UK). Again polymer molecular weights quoted are those claimed by the manufacturer. The manufacturers designated code was used throughout the study. Yttrium zirconia beads of size 0.65 mm (range 0.55–0.69 mm) and 0.44 mm (0.35–0.5 mm range) were obtained from the Nikkato Corp. (Tokyo, Japan). Spectroscopically pure water (18.2 MV) obtained from an Elga Maxima ultra
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pure water unit (High Wycombe, UK) was used for studies of polymer adsorption; distilled water was used in all other experiments. All chemicals were used as received and polymer concentrations were not corrected for any moisture that may be present.
Molecular Weight Characterisation of Polymers An estimate of the viscosity average molecular weight (Mv) of the HPMC and PVP polymers were obtained from capillary viscometry. The amount of polymer used to prepare the stock solutions varied with the molecular weight of the polymer to ensure that measurements were made below C (defined as 1/[h] where [h] is the intrinsic viscosity of the polymer). Polymer solutions were prepared at 5% w/v (PVP 12), 1.0% w/v (PVP 30), 1.2% w/v (HPMC 8), 1.0% w/v (HPMC 11), 0.25% w/v (HPMC 24), 0.1% w/v (HPMC 86) in distilled water. Automated measurements of flow times of the polymer solutions in an Ubbelholde viscometer were carried out using a Viscosity Measuring Unit AVS 350 (Schott-Gera¨te, Hofheim, Germany), connected to a ViscoDoser AVS 20 piston burette (for automatic dilutions). The viscometer was immersed in a precision water bath (transparent thermostat CT 1650, SchottGera¨te, Hofheim, Germany) to maintain the temperature at 20 0.18C. Flow time measurements of each polymer solution were made in triplicate. The intercept of separate Huggins and Kramer extrapolations (linear regression) yielded [h] and from this the molecular weight was determined by applying the Mark-Houwink equation for HPMC with the constants K and a being set to 3.39 104 and 0.88.18 The molecular
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weights (Mv) of the HPMCs used in this study were found to range between 5 and 89 kg/mol (Table 1). For PVP, the K-value (i.e., the Fikentscher K-value, not the constant K of the Mark-Houwink equation) was calculated for each PVP using the equation given in the British Pharmacopeia.21 From the K-value it was possible to calculate the viscosity-average molecular weight (Mv).22 The results of this analysis are given in Table 1. For both types of polymer the results of the viscosity and molecular weight determination are expressed as the mean the standard deviation of three individual measurements. The number (Mn) and weight (Mw) averaged molecular weight of PVP 12, 30, and 90 and of HPMC 8 and HPMC 86 were determined using gel permeation chromatography (GPC) by Rapra Technology (Shrewsbury, UK) (Table 1). For PVP, a solution of the sample was prepared by dissolving 25 mg in 15 cm3 dimethylformamide (DMF). The solution was filtered through a 0.2 mm polyamide membrane into sample vials and placed in an autosampler. GPC was carried out on a Waters 150 CV instrument using a Plgel 2 mixed bed-B, 30 cm, 10 mm column (Polymer Laboratories, Shropshire, UK) using DMF containing 0.01 M lithium bromide as the solvent, at a flow rate of 1.0 cm3/min (nominal) at 808C with a refractive index detector. The data collected was analysed using Viscotek Trisec 3.0 software (Table 1). This method was used in the present study as it has been previously found23 to yield PVP molecular weights that were in good agreement with those obtained by static light scattering measurements. For HPMC, a solution of polymer was made by dissolving in a 0.2 M aqueous
Table 1. Intrinsic Viscosity [h] at (208C) and Viscosity-Average Molecular Weight (Mv) from Capillary Viscosity Measurements (n ¼ 3 s.d.) and Number (Mn) and Weight-Average (Mw) Molecular Weight and Polydispersity ( Pd ¼ Mw/Mn) of Polymer Solutions from GPC (mean, n ¼ 2)
Capillary Viscometry Polymer Identification HPMC 8 HPMC 11 HPMC 24 HPMC 86 PVP 12 PVP 30 PVP 90
GPC
Nominal Molecular Weight (kg/mol)a
[h] (mL/g)
Mv (kg/mol)
Mw (kg/mol)
Mn (kg/mol)
Pd
8 11 24 86 3.9 42.5 1300
61.4 0.2 88.9 1.2 306.2 1.7 770.1 2.9 4.7 0.1 20.8 0.1 N/D
5.0 0.0 7.7 0.1 31.3 0.2 89.1 0.4 3.0 0.1 46.2 0.4 N/D
23.1 45.6 298.0 687.5 3.4 41.0 560
4.2 5.8 44.0 56.2 2.7 16.0 140
5.5 7.9 6.8 12.0 1.2 2.6 4.0
N/D ¼ not determined. a Molecular weight as stated by manufacturer. DOI 10.1002/jps
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solution of ammonium acetate. The resulting solution was filtered through a 0.45 mm PVDF filter directly into sample vials and placed in an autosampler prior to GPC analysis using two Plgel mixed-OH, 30 cm, 8mm columns fitted with a Plgel guard column and a refractive index detector. The GPC analysis was performed at a temperature of 30oC and flow rate of 1.0 cm3/min was employed. A summary of the number (Mn) and weightaverage (Mw) molecular weights determined in this way as well as the polydispersity ( Pd) of the polymers are given in Table 1. The results are expressed as the mean of two separate measurements. In no case did the duplicate measurements deviate by more than 10%.
Production of Nabumetone and Halofantrine Nanoparticles Preparation of Crude Suspensions Polymer solutions were prepared by weighing the appropriate amount of polymer and making to weight with distilled water. Once the polymer had dissolved, sufficient polymer solution was added to either 10 g of nabumetone or 15 g of halofantrine with constant (magnetic) stirring to form 50 g of crude suspension or slurry. It should be noted that the polymer concentrations are generally quoted in the text as the weight % of polymer in the solution, which was added to drug to form a crude suspension prior to milling and not the final concentration in the crude drug suspension.
nabumetone suspension with 1.88% w/w HPMC 8 (NB the wet-bead milling process requires a high drug loading). Varying volumes of beads and crude suspension were added to a 25 cm3 volume milling jar made from food-grade nylon (Nylacast, Leicester, UK) and milled for 6 h using a Retsch MM200 Mixer Mill (Glen Creston, Stanmore, UK) at 30 vibrations/s. For each condition studied three separate suspensions were prepared and milled and, therefore, the results are expressed as the mean of three measurements the standard deviation (Table 2). ‘‘Optimum’’ process parameters of 10 cm3 of 0.44 mm beads and 10 cm3 of crude drug suspension were selected on the basis of being the protocol which yielded the minimum nanoparticle size obtained as assessed by laser diffraction after 6 h milling; milling for longer periods did not appear to cause any further significant reduction in particle size. These parameters were used for the milling of all subsequent formulations.
Physical Characterization of Nanoparticles Particle Size Analysis: Laser Diffraction
Wet-Bead Milling
Particle size analysis of nanoparticles was performed using a Malvern 2600 series laser diffractometer (Malvern Instruments, Malvern, UK). Nanosuspensions were diluted in distilled water to obtain an acceptable obscuration level for size determination. From the particle size data, the volume (or mass) moment mean (D[4,3]) was used to characterize the nanoparticles (Eq. 1). P 4 d D½4; 3 ¼ P 3 (1) d
A preliminary study into the effect of bead loading, slurry (crude suspension) loading, and bead size was undertaken using a 20% w/w
The volume moment mean was chosen for this study as it is sensitive to the presence of large
Table 2. Effect of Slurry and Bead Loading and Bead Size on the Mean Particle Size of nabumetone after 6 h of wet-bead milling in the presence of 1.88% w/w HPMC 8 (n ¼ 3 s.d.)
Bead Loading (mL)
Slurry Loading (mL)
Bead Size (mm)
Mean Particle Sizea (mm)
12 8 7 10 12 8 7 10
0.44 0.44 0.44 0.44 0.65 0.65 0.65 0.65
0.79 0.04 0.74 0.00 0.69 0.06 0.65 0.06 0.79 0.04 0.75 0.01 0.77 0.03 0.76 0.01
7 7 12 10 7 7 12 10 a
Obtained from laser diffraction.
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particles in the sample24 and allows us to establish whether any large particles are still present in the nanosuspension because even if they are small in terms of their number, they would make up a considerable mass of the drug particles. (It should be mentioned that, because of the polydispersity of the nanoparticles and the assumption of spherical particles, the mean size of the particles determined depends upon the precise technique used to measure particle size, for example, use of photon correlation spectroscopy (Brookhaven ZetaPlus Sizer 2.29) yielded nanoparticle sizes about half that obtained using laser diffraction). Electron Microscopy A drop of milled nanosuspension was placed on the surface of a small (ca. 5 5 mm) piece of freshly cleaved mica and allowed to dry. This was then mounted onto a specimen stub using double-sided adhesive tape, and coated with a layer of gold approximately 15 nm thick using a Polaron E5100 sputter coater. A Philips SEM501B scanning electron microscope operating at 15 kV was used to visualize the samples. Freeze fracture microscopy was carried out on nanosuspensions of 20% w/w nabumetone prepared using a 1.25% w/w solution of HPMC 8, 30% w/w halofantrine with 1.43% w/w HPMC 8 and 30% w/w halofantrine prepared with 1.43% w/w PVP30. Suspensions were prepared according to the method described above. Subsequent to 6 h wet-bead milling, the nanoparticle suspensions were subject to size analysis and then centrifuged at 13,000 rpm for 1.5 h. Determination of Polymer Concentration A series of solutions were prepared containing 0.01, 0.1, 1, 3, and 5% w/w of either HPMC 8, HPMC 11, or HPMC 24 in ultra-pure water. The UV spectra of the solutions were obtained over a wavelength range of 180–600 nm on a p180 spectrophotometer (Applied Photophysics Ltd., Leatherhead, UK.). The instrument was continually flushed with nitrogen. All samples, including ultra-pure water used in the preparation of standard samples (prepared and measured in triplicate) were scanned against air and the absorbance of water subtracted from that of the HPMC containing samples in order to obtain the UV spectra of HPMC. Optical rotatory dispersion (ORD) spectra were obtained using a Jasco J600 spectropolarimeter (Jasco UK Ltd., DOI 10.1002/jps
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Great Dunmow, UK) with a spectral bandwidth of 2 nm, scanning rate of 20 nm/min and data resolution of 1 nm. Solutions to be measured were placed in a 5 cm cell and the ORD measured over a wavelength range of 650–230 nm. The values of the ORD at 350 nm were plotted as a function of HPMC concentration. The assay for PVP was based on UV spectroscopy. A range of solutions of different PVPs namely PVP 12, 30, and 90 were prepared from a stock solution in spectroscopically pure water. Each dilution was prepared at least in triplicate. The solutions were scanned in a cuvette with 0.1 cm pathlength over a wavelength range of 185–400 nm using a p180 spectrophotometer with a spectral bandwidth of 2 nm, at a scanning rate of 50 nm/min and a step size of 1 nm. All samples, including the water used to prepare the samples were scanned against air and the absorbance of water subtracted from that of the PVP containing samples. The UV absorbance at a wavelength of 195 nm was plotted as a function of concentration. Determination of Polymer Adsorption The amount of HPMC 8 adsorbed onto nabumetone nanoparticles was obtained by measuring its depletion from solution using the following procedure. Ten cubic centimeter of aqueous suspensions comprising 20% w/w nabumetone and various concentrations of HPMC 8 were milled for 6 h using a Restch MM200 mill with 10 cm3 of 0.44 mm beads. The resulting nanosuspensions were then centrifuged at 13,000 rpm for 1.5 h. The supernatants were collected and assayed for HPMC content by measuring the ORD spectrum over 650–230 nm using a 2 cm path length cell. A smaller path length cell than was used for calibration purposes was necessitated for examining the test sample due to the low volume of polymer solution collected after removal of the drug and adsorbed polymer. Furthermore, due to the low volume of nanosupsensions produced after milling, the ORD of each test sample could only be measured once. Therefore, three individual nanosupensions were prepared and the ORD of each measured so that the concentration of HPMC 8 remaining in solution after adsorption could be expressed as the mean s.d. of three determinations. By determination of the concentration of polymer remaining in the supernatant, it is possible to calculate the amount of polymer lost from solution
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and thereby determine the amount of polymer adsorbed onto the drug. In order to determine the amount of polymer adsorbed an estimate of the total surface area of the drug nanoparticles was made based on particle size analysis by laser diffraction, assuming the drug particles to be spherical, and using the density of the drug determined using a StereopycnometerTM (Quantachrome Instruments, Hook, UK). The pycnometer uses Archimede’s principle of fluid (gas) displacement and the technique of gas expansion. In the present study, helium was used as the displacing fluid (gas). The density of nabumetone was determined to be 1.19 g/cm3 and halofantrine to be 1.39 g/cm3. The same methodology was repeated to obtain adsorption isotherms for halofantrine with HPMC 8 or PVP 30. However, the amount of PVP left in solution after the adsorption was complete was quantified using UV spectroscopy as described above.
RESULTS AND DISCUSSION Effect of Polymer and Concentration of Nanoparticle Production Size analysis of nabumetone particles after 6 h milling in the presence of HMPC shown in Table 3. Note that nabumetone nanoparticles could not be prepared if PVP was used as stabling polymer. Also given in Table 3 is the weight ratio of drug:polymer used in the present study. This
value is reported in the present study because it has been stated by other workers, for example, Merisko-Liversidge et al.9 and Date and Patravale,25 as being an important factor when preparing nanoparticles. These workers quote, respectively, that a drug:stabilizer weight ratio in the range of 20:1 to 2:1 or a maximum ratio of drug:stabilizer of 1.7:1 as being necessary for nanoparticle production. Although the weight ratio of drug:stabilizer used for nanoparticle production might be a useful parameter for the formulator, the present authors do not find it particularly helpful in understanding the mechanism of polymer adsorption since it neglects amongst other things the density of the drug, the size of the nanoparticles, and the molecular weight of the polymer. Furthermore, as can be seen from Table 3 there are a number of instances in the present study where nanoparticles were produced using weight ratios of drug:polymer outside the quoted range. From Table 3, it is clear that for each polymer molecular weight that a minimum and maximum concentration of HPMC is required to produce and stabilize the nabumetone nanoparticles. At HPMC concentrations below the minimum required for production of nabumetone nanoparticles, it is probable that there is insufficient polymer present to completely cover the surface of the drug particle in order to provide a steric barrier against the cohesive forces between the drug particles that lead to the aggregation and agglomeration of these small sized particles. At the other end of the concentration range, at
Table 3. Effect of Polymer Molecular Weight on the Mean Particle Size of Nabumetone Milled for 6 h in the Presence of Various Concentrations of HPMC (n ¼ 3 s.d.)
Nominal HPMC Molecular Weight (g/mol)/Mean Particle Size of Nabumetone after 6 h Bead Milling (mm) HPMC Concentration (% w/w) 0.06 0.13 0.63 0.94 1.25 1.56 1.88 2.50 3.75 6.25
Weight Ratio of Drug to Polymer
HPMC 8
HPMC 11
HPMC 24
HPMC 86
417:1 192:1 40:1 27:1 20:1 16:1 13:1 10:1 7:1 4:1
17.80 1.03 2.96 0.07 0.75 0.01 0.86 0.02 0.65 0.04 0.87 0.01 0.65 0.4 0.90 0.01 1.27 0.29 1.81 0.88
ND 6.93 3.52 0.89 0.03 0.74 0.01 0.86 0.01 0.96 0.03 0.78 0.01 1.16 0.04 2.42 0.07 ND
ND 3.32 0.12 2.19 0.12 2.19 0.03 0.94 0.01 1.61 0.04 1.98 0.19 ND ND ND
18.69 9.04 11.51 1.39 2.67 0.15 2.31 0.04 Solid Solid Solid Solid Solid Solid
Solid ¼ suspension solidified in the milling jar after milling. ND ¼ not determined. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 96, NO. 10, OCTOBER 2007
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stabilizer levels above the maximum HPMC concentration that produces nabumetone nanoparticles, the average size of the drug particles produced by milling increased to above 1 mm and, therefore, the anticipated benefits in dissolution rate of the nanoparticles lost. As can be seen from Table 3, the concentration range over which HPMC could produce nanoparticles was found to decrease with an increase in the molecular weight of the polymer. For example, using HPMC 24, nabumetone nanoparticles could only be produced at a concentration of 1.25% w/w whereas it was not possible to produce nanoparticles at any of the concentrations studied when using HPMC 86 as the stabilizing polymer. The reduction observed in the maximum concentration of polymer that can be used to produce nanoparticles as the molecular weight of the stabilizing polymer is increased is thought to be, at least in part, a consequence of the accompanying increase in the viscosity of the suspension. It has been suggested that since the milling process relies upon the impaction of beads on drug particles increasing the viscosity of the suspension being milled may lead to the beads traveling at a lower velocity, thereby resulting in smaller forces of impaction and, therefore, less efficient size reduction.26,27 This hypothesis is in agreement with simulations of the motion of balls in a vibration mill performed by Yokoyama et al.28 in which an increase in the viscosity of the surrounding medium caused a radical drop in the intensity of ball collisions. The observation that the minimum concentration of HPMC required for nanoparticle production also increases with the higher molecular weight grades can be partly explained by a combination of the slow diffusion of the higher molecular weight polymers to the ‘‘bare’’ drug surfaces created during milling and the occurrence of bridging flocculation at these relatively low polymer concentrations. Bridging flocculation is where a single polymer molecule is adsorbed to more than one particle.29 The inability of the higher molecular weight polymers to prepare nanoparticles may have important implications for the stability of the resulting nanoparticles as it is well established that higher molecular weight polymeric stabilizers tend to be favored since they are reported to yield a more stable dispersion compared to lower molecular weight grades of the same polymer. This is generally attributed to the fact that higher molecular weight polymers form a thicker adsorbed layer and thus offer greater DOI 10.1002/jps
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stabilization upon approaching particles than lower molecular weight polymers.30 By way of comparison between the two drugs studied, when halofantrine was milled in the presence of HPMC 8 and HPMC 24; nanoparticles could be formed between 0.71% w/w and 2.14% w/ w HMPC 8 (HPMC 8 concentrations outside this range were not examined) and at 0.14% w/w HPMC 24 (surprisingly nanoparticles were not produced at HPMC 24 concentrations of 0.71 and 1.43% w/w). Unfortunately, due to a limited supply of halofantrine it was not possible to perform the experiments needed to allow the upper and lower limit of stabilizing polymer concentration to be obtained. Significantly, however, it was not possible to prepare nabumetone nanoparticles using only 0.14% w/w HPMC 24, which suggests a different mechanism of coating of the two types of nanoparticles by HPMC, especially bearing in mind the higher drug loading/lower amount of polymer present in the halofantrine preparation. In contrast to nabumetone (data not shown), it was also found possible to produce halofantrine nanoparticles using either PVP 12 or PVP 30 as a stabilizer in the range 0.71–2.14% w/w, although the upper and lower concentration limits were not found due to constraints on the amount of halofantrine available. It is worth commenting that the nanoparticles prepared using the present methodology were stable with respect to size when stored at 258C for at least 1 year, longer periods were not studied (data not shown). No evidence of Ostwald ripening was seen in any of the nanosuspensions tested. This observation agrees with that of MeriskoLiversidge et al.9 who reported that in the liquid state such polymer stabilized nanoparticles were very stable, especially if the solubility of the drug was less than 1 mg/cm3 (as in the present study). Furthermore, the nanoparticles were stable regardless of the concentration at which the nanoparticles were stored, that is, whether they were stored at the concentration at which they were prepared or stored in a diluted form. In addition, the nanoparticles could be readily resuspended after centrifugation and replacement of the excess polymer solution with an equal volume of water; the nanoparticles in this state were also stable for periods of at least 1 year. All nanosuspensions, however, did exhibit sedimentation upon standing, although as expected, the sedimentation rate in the nanosuspensions containing excess polymer was slower than in its absence. In addition, HPMC stabilized
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nanoparticles remained uniformly dispersed for longer than the PVP-stabilized ones, which tended to form a sediment which was harder to redisperse. Morphology of Nanoparticles The SEM images of nabumetone nanoparticles milled for 6 h in the presence of 1.25% w/w HPMC 8 (Fig. 1) and 1.25% w/w HPMC 24 (Fig. 2). As can be seen in Figures 1 and 2 the majority of nanoparticles were polydisperse rods. Reassuringly an estimate of the size of the nabumetone nanoparticles based on the SEM images confirms the general findings from laser diffraction size analysis, with the nanoparticles prepared using HPMC 24 exhibiting the slightly larger size. It is worth commenting that the somewhat melted appearance of the nabumetone nanoparticles is thought to be a consequence of the nanoparticles melting in the vacuum of the electron microscope (the melting point of nabumetone at 1 atmosphere is 78–838C). Figure 3 shows the SEMs obtained of halofantrine nanoparticles milled in the presence of either 1.43% w/w HPMC 8 or 1.43% w/w PVP 30. As can be seen the halofantrine nanoparticles milled in the presence of HPMC 8 appeared more irregular in shape than the rod-shaped nabumetone nanoparticles prepared using HPMC 8 (Fig. 1). In addition, they varied from the halofantrine nanoparticles formed in the presence of PVP 30, which appeared cuboidal at larger sizes, and spherical at smaller sizes, possibly due to the
Figure 2. SEM of nabumetone suspension milled in the presence of 1.25% w/w HPMC 24 for 6 h.
attrition of the hard edges of the particle as milling progresses. According to Merisko-Liversidge et al.,9 the geometrical shape of milled drug nanoparticles is dependent upon the morphology of the starting drug material, the fracture plane of the crystals and drug/stabilizer interactions. Although the influence of the original shape of the drug crystal in determining the final shape of the nanoparticles has not been examined in the present study, it does appear that the type of stabilizer used does have an affect on the shape of the resulting nanoparticles based on halofantrine nanoparticle SEM images. Adsorption Isotherms
Figure 1. SEMs of nabumetone suspension milled in the presence of 1.25% w/w HPMC 8 for 6 h.
Quantification of the amount of polymer adsorbed onto the drug nanoparticles was made using the depletion method. This method involves determining the concentration difference of polymer in the continuous phase before and after milling and relating this to the surface area of drug available for adsorption. The available surface area of the drug is estimated by determining the number and size of the nanoparticles from the laser diffraction studies assuming spherical nanoparticles. Unfortunately, in the present study, the nonspherical polydisperse nature of the nanoparticles (as seen in the SEMs of both nabumetone and halofantrine nanoparticles, Figs. 1–3) will introduce a degree of error into this calculation. In order to estimate how much polymer has adsorbed onto the drug, a suitable analytical technique is required for the adsorbate.31
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Figure 3. SEM images of halofantrine nanoparticles comparing the effect of the stabiliser on nanoparticle morphology (a) halofantrine nanoparticles prepared with 1.43% w/w HPMC 8 and (b) halofantrine nanoparticles prepared with 1.43% w/w PVP 30.
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Although colourimetric assays are available for both PVP32 and HPMC (USP23 assay of Hypromellose Ophthalmic Solution),33 such assays are very dependent upon the molecular weight of the polymer being assayed, particularly when using relatively low molecular weight polymers such as those used in the present study. The nonmolecular weight dependence of the assay is essential in the present study because fractionation of the polymer is reported to occur at the solid–liquid interface due to the preferential adsorption of longer polymer chains.34 In the light of these findings, an ORD assay was developed for HPMC and UV spectroscopy was used to quantify the concentration of PVP in solution. The UV assay developed was independent of polymer molecular weight over the nominal weight range of 3.9– 1,300 kg/mol (data not shown). A critical requirement for nanoparticle production is that the stabilizing agent adsorbs to the surface of the drug particle to prevent agglomeration.7 Establishment of the adsorbed amount is, therefore, an important parameter to characterize polymer-stabilized nanoparticles, the extent of adsorption being based on competing interactions between the drug particle surface, the polymer, and the solvent molecules.35 The dependence of the amount of polymer adsorbed onto the drug after 6 h of milling on the initial concentration of HPMC 8 is given in Table 4. As can be seen from Table 4, the profile of the adsorption isotherm is different to those commonly reported in the literature31 since the initial (rising) portion of the isotherm is absent due to the fact that nanoparticles can only be produced once the concentration of stabilizer is above a certain threshold in order to completely coat the surface of the drug particle, discussed above. At a starting concentration of 0.63% w/w HPMC 8, it can be seen from Table 4 that a polymer concentration of 0.17% w/w was left in solution, so that as a consequence 76 mg of HPMC 8 was adsorbed on to 4 g of
Table 4. Mean Adsorption of HPMC 8 on Milled Nabumetone Particles at Various Concentrations (n ¼ 3 s.d.)
Starting Polymer Concentration (%w/w) 0.63 0.94 1.25 1.56 1.88 DOI 10.1002/jps
Mean Concentration of Polymer in Solution after Adsorption (%w/w)
Mean Amount of Polymer Adsorbed onto Drug (mg)
Mean Amount of Polymer Adsorbed onto Drug (mg/m2)
0.17 0.08 0.31 0.15 0.55 0.25 1.20 0.25 1.16 0.10
76.0 14.0 105.4 24.7 115.9 8.4 59.9 42.0 119.2 17.0
2.8 0.5 4.5 1.1 3.7 0.3 2.6 1.8 3.8 0.6
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Table 5. Mean Adsorption of HPMC 8 on Milled Halofantrine Particles at Various Concentrations (n ¼ 3 s.d.)
Starting Polymer Concentration (%w/w) 0.71 1.07 1.43 1.79 2.14
Mean Concentration of Polymer in Solution after Adsorption (%w/w)
Mean Amount of Polymer Adsorbed onto Drug (mg)
Mean Amount of Polymer Adsorbed onto Drug (mg/m2)
0.09 0.05 0.35 0.02 0.81 0.02 1.08 0.23 1.44 0.19
97.8 7.9 112.9 2.7 96.7 3.6 111.4 35.3 109.8 29.1
2.5 0.2 2.8 0.1 2.7 0.1 2.7 0.9 3.1 0.8
nabumetone nanoparticles. This value represents the minimum amount of polymer required to stabilize nabumetone nanoparticles. Particle size analysis results (Table 3) confirm this finding since milling of nabumetone with a concentration of HPMC less than 0.46% w/w, for example, 0.13% w/w, led to the production of micro- rather than nanoparticles. By making the assumptions described above, it is possible to determine the mg of drug absorbed per m2 of drug. It can be seen from Table 4 that increasing the starting concentration of HPMC had little effect on the amount of polymer adsorbed, suggesting that the absorption plateau had been reached. It is worth noting that the adsorbed amount of polymer is much larger than is normally quoted in the literature for comparable systems, e.g. polymer adsorption on polystyrene nanoparticles. This discrepancy is undoubtedly partly (not but not solely) due to the assumption of monodisperse spherical particles, which will underestimate the surface area of drug available for absorption. The adsorption of HPMC 8 on halofantrine (Table 5) resulted in a slightly lower plateau value than HPMC 8 on nabumetone and may be attributed to a difference in affinity of HPMC 8 for the surface of the two drugs. A lower adsorption of PVP 30 was found on halofantrine (Table 6) compared with HPMC 8 suggesting that smaller amounts of PVP 30 than HPMC 8 are required to stabilize the halofantrine nanoparticles. As both
HPMC and PVP are nonionic polymers and the two drugs studied are neutral species, the forces of attachment of these polymers onto the surface of the drugs can be attributed to either hydrophobic interactions or via hydrogen bonding.36 In order to ensure that the assumptions used in estimating adsorbed amount did not alter the trends of the results, calculations were also performed assuming the presence of nanoparticles of equivalent volume to the spheres measured using laser diffraction but approximating the shape of nanoparticles to that seen using FFEM, namely cubes for halofantrine nanoparticles prepared in the presence of PVP 30 and rodlike particles (length to width ratio of 3:1) for both nabumetone and halofantrine nanoparticles milled with HPMC 8. As the milled nanoparticles are not regular in shape this approach is also in error but serves to illustrate that the differences observed between the different combinations of drug and polymer are genuine and not due to the assumptions of nanoparticle shape made in the present study. As would be anticipated (from surface area:volume considerations), the amount absorbed was greatest assuming spherical particles (minimum surface area to volume) was 20% less when cuboids were assumed and 28% less when rodlike particles were assumed. While the amount adsorbed obviously varied depending upon the shape of the nanoparticles assumed, the trend of the results obtained assuming
Table 6. Mean Adsorption of PVP 30 on Milled Halofantrine Particles at Various Concentrations (n ¼ 3 s.d.)
Starting Polymer Concentration (%w/w) 0.71 1.07 1.43 1.79 2.14
Mean Concentration of Polymer in Solution after Adsorption (%w/w)
Mean Amount of Polymer Adsorbed onto Drug (g)
Mean Amount of Polymer Adsorbed onto Drug (mg/m2)
0.46 0.02 0.75 0.06 1.18 0.07 1.48 0.05 1.92 0.08
39.2 3.1 50.2 9.4 39.2 11.0 48.6 7.8 34.5 12.6
1.0 0.1 1.4 0.3 1.2 0.3 1.4 0.2 0.9 0.3
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spherical nanoparticles described below does not: with cuboidal halofantrine nanoparticles milled in the presence of PVP 30 exhibiting about a thirdhalf of the absorbed amount of rodlike nabumetone and halofantrine nanoparticles milled in the presence of HPMC 8. The differences in adsorbed amount recorded for the different drug/polymer combinations may reflect differences in the conformation of the adsorbed polymer on the surface of the drug nanoparticles. These differences may be due to the differing nature of the two polymers and/or differences in polymer molecular weight (Table 1). Further work such as small angle neutron scattering studies is required to determine the conformation of the polymer on the surface of the drug nanoparticles.
CONCLUSION Higher molecular weight polymers tend to be favored for steric stabilization since they are reported to yield a more stable dispersion compared to lower molecular weight grades of the same polymer. As the method of producing nanoparticles using wet-bead milling demands a high drug loading, this precludes the use of high molecular weight polymeric stabilizers due to the resulting high viscosity of the milling slurry. However, in the present study it has been found that the molecular weight of the polymer used did not influence drug nanoparticle stability.
ACKNOWLEDGMENTS The authors would like to acknowledge the help of Professor SM Ross-Murphy and Dr. D Picout in performing the viscosity measurements, and Dr. T Bui in developing the analytical technique for assay of PVP and HPMC. The authors also express their appreciation to Dr. A Brain at the KCL Electron Microscopy Unit for his assistance in obtaining the SEM and FFEMs. In addition, SS and DG thank EPSRC, Impact Faraday and GSK for the award of studentships.
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