pubs.acs.org/Langmuir © 2009 American Chemical Society
Scanning Probe Microscopy Method for Nanosuspension Stabilizer Selection Sudhir Verma,† Bryan D. Huey,‡ and Diane J. Burgess*,† †
Department of Pharmaceutical Sciences and ‡Department of Chemical, Materials and Biomolecular Engineering, University of Connecticut, Storrs, Connecticut 06269 Received May 8, 2009. Revised Manuscript Received June 12, 2009 An atomic force microscopy (AFM) method was successfully developed and utilized for investigating the interaction of polymeric stabilizers with ibuprofen to determine their suitability for the preparation and stabilization of ibuprofen nanosuspensions. Images obtained clearly showed that HPMC and HPC interacted strongly with the ibuprofen resulting in extensive surface adsorption, confirming their suitability for ibuprofen nanosuspension preparation. In addition, differences in the morphology of the adsorbed HPMC and HPC molecules were observed, which may be attributed to their variable degree of substitution. Consistent with their poor performance in stabilizing the ibuprofen nanosuspensions, images obtained with PVP and Poloxamer’s depicted inadequate adsorption on the ibuprofen surface. Careful analysis of the AFM images and the ibuprofen crystal structure gave valuable insight into the success of topdown processing for the preparation of nanosuspensions as compared to bottom-up processing. On the basis of the relationship observed between nanosuspension stability and adsorption characteristics of specific polymers, such AFM studies can aid in the selection of suitable nanosuspension stabilizers. This method provides the basis for a scientific rationale for nanosuspension stabilizer selection rather than the trial and error method which is currently practiced.
Introduction Nanosuspensions are defined as colloidal dispersions of discrete drug particles with particle size in the range 1-1000 nm. They are being actively pursued for the formulation of pharmaceutical compounds with poor aqueous solubility. The small size and high surface area of drug nanosuspensions result in increased dissolution rates and hence improved bioavailability of water insoluble compounds and thus can overcome the basic hurdle of poor dissolution rate in the successful development of these compounds. Besides improved bioavailability,1,2 altered disposition,3 increased chemical stability,4 increased drug loading,5 and reduced toxicity and side effects6 are additional biopharmaceutical advantages of the nanosuspensions. Any nanosuspension formulation has three basic ingredients: active pharmaceutical ingredient (API), stabilizer, and dispersion medium. Usually, the dispersion medium is water, and the API is a hydrophobic drug compound with poor aqueous solubility. The stabilizers are surface active agents or polymers that adsorb at the interface of the drug particles with water. Stabilizers commonly used to stabilize nanosuspensions include polymers (such as polyvinyl pyrrolidone (PVP), hydroxypropyl methyl cellulose (HPMCs), and hydroxypropyl cellulose (HPCs)), ionic surfactants (e.g., sodium dodecyl sulfate (SDS)), and nonionic surfactants (e.g., Tweens and Poloxamers (polyoxyethylene-polyoxypropylene copolymers)). Ionic surfactants stabilize suspensions via electrostatic repulsion, while polymers and nonionic surfactants facilitate suspension stability via steric repulsion. *Corresponding author. Diane J. Burgess. Phone: 860-486-3760. Fax: 860486-0538. E-mail:
[email protected].
(1) Liversidge, G. G.; Cundy, K. C. Int. J. Pharm. 1995, 125, 91–97. (2) Kraft, W. K.; Steiger, B.; Beussink, D.; Quiring, J. N.; Fitzgerald, N.; Greenberg, H. E.; Waldman, S. A. J. Clin. Pharmacol. 2004, 44, 67–72. (3) Yeh, T. K.; Lu, Z.; Wientjes, M. G.; Au, J. L. S. Pharm. Res. 2005, 22, 867– 874. (4) Moschwitzer, J.; Achleitner, G.; Pomper, H.; Muller, R. H. Eur. J. Pharm. Biopharm. 2004, 58, 615–619. (5) Rabinow, B. E. Nat. Rev. Drug Discovery 2004, 3, 785–796. (6) Liversidge, G. G.; Conzentino, P. Int. J. Pharm. 1995, 125, 309–313.
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The high surface area of drug nanoparticles, by the virtue of which they exhibit their unique biopharmaceutical characteristics, also renders them thermodynamically unstable5 and promotes agglomeration and crystal growth. This high surface area increases the total free energy (ΔG = γs/l* ΔA) of the system, where ΔA is the total surface area of the particles and γs/l is the interfacial tension between the drug surface and aqueous phase. Consequently, stabilizers are an indispensible component of nanosuspension formulations, since they adsorb at the interface, reducing the interfacial tension (γs/l) and thereby decreasing the total free energy ΔG (ΔG = γs/l* ΔA) of the system. Stabilizers play a crucial role in the formation, stability, and overall performance of nanosuspensions.7,8 Both nanoparticle creation and subsequent stabilization are highly sensitive to the choice of stabilizer. Paradoxically, the current practice used to select stabilizers is a trial and error approach with no prior knowledge of their ability to interact with the drug surface.9,10 Recently, a few studies focused on developing empirical relationships between stabilizer efficacy and some property of the drug compound have been published to aid in stabilizer selection. In one such study, Choi et al.11 investigated the stabilizing efficiency of polymers as a function of similarity between the surface energies of the polymer and the drug. They concluded that, though comparing surface energies may provide some help, specific interactions between the stabilizer and the drug appeared to play a more important role in the formation of the nanosuspensions. Lee et al.7 prepared nanosuspensions of several water insoluble drugs with various polymers in an attempt to establish a general guide for the preparation of nanosuspensions. A rule of (7) Lee, J.; Choi, J. Y.; Park, C. H. Int. J. Pharm. 2008, 355, 328–336. (8) Eerdenbrugh, B. V.; Vermant, J.; Martens, J. A.; Froyen, L.; Humbeeck, J. V.; Augustijns, P.; Guy Van den Mooter, G. V. D. J. Pharm. Sci. 2008, 98, 2091– 2103. (9) Jacobs, C.; Muller, R. H. Pharm. Res. 2002, 19, 189–194. (10) Lee, J.; Lee, S. J.; Choi, J. Y.; Yoo, J. Y.; Ahn, C. H. Eur. J. Pharm. Sci. 2005, 24, 441–449. (11) Choi, J. Y.; Yoo, J. Y.; Kwak, H. S.; Nam, B. U.; Lee, J. Curr. Appl. Phys. 2005, 5, 472–474.
Published on Web 09/30/2009
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thumb was proposed that drugs with high molecular weights, low solubility, high melting points, and surface energies similar to that of the stabilizer used could be successfully processed into nanosuspensions. Similarly, Eerdenbrugh et al.8 investigated the formation of nanosuspensions of several drugs with various polymeric stabilizers. In this study, it was concluded that the hydrophobicity of the drug played a significant role in the production of the drug nanoparticles. However, none of these studies afford adequate assistance in the systematic selection of appropriate stabilizers for nanosuspensions. Thus, the key challenge remains to develop a screening method for nanosuspension stabilizer selection. The aim of this study was to investigate local interactions between the stabilizer and the drug surface to determine whether this may assist formulation scientists in the judicious choice of stabilizers. Atomic force microscopy (AFM) was employed for the first time to investigate drug-stabilizer interactions through visualization of stabilizers adsorbed directly onto the drug surface. Although AFM has been used successfully in the past for the imaging of soft structures such as adsorbed surfactant and polymers onto solid substrates in both gaseous and liquid environments, all measurements were performed with substrates easily acquired with a nearly atomically flat surface (e.g., mica, silica, or graphite).12-18 Direct visualization of the adsorbed stabilizer directly on the drug surface will therefore not only aid in comprehending the extent of interaction between the drug and the stabilizer, but also afford vital information about the characteristics of the adsorbed layer such as the specific arrangement of the stabilizer molecules, the thickness of the adsorbed layer, and the strength of the interfacial film. All of these characteristics are known to significantly affect wettability,19 interparticulate interactions,20-24 crystal growth, Ostwald ripening, and aggregation.25-27 A comprehensive knowledge on specific drug-stabilizer interactions and other fundamental details about the exact nature of the interfacial adsorbed layer will thus pave the way toward a more scientific basis for stabilizers selection.
Materials and Methods Materials. Ibuprofen USP, 2-[4-(2-methylpropyl) phenyl] propanoic acid was purchased from PCCA (Houston, TX). Methocel (hydroxypropyl methylcellulose) E5 premium LV grade was generously gifted by Dow Chemical Company (Midland, MI). Poloxamer 188 (Pluronic F-68), Poloxamer 407 (Pluronic F-127), and Kollidon 30 (PVP K-30) were purchased from BASF (12) Arita, T.; Kanda, Y.; Higashitani, K. J. Colloid Interface Sci. 2004, 273, 102–105. (13) Ducker, W. A.; Grant, L. M. J. Phys. Chem. 1996, 100, 11507–11511. (14) Fleming, B. D.; Wanless, E. J. Microsc. Microanal. 2000, 6, 104–112. (15) Fleming, B. D.; Wanless, E. J.; Biggs, S. Langmuir 1999, 15, 8719–8725. (16) Grant, L. M.; Ducker, W. A. J. Phys. Chem. B 1997, 101, 5337–5345. (17) Grant, L. M.; Tiberg, F.; Ducker, W. A. J. Phys. Chem. B 1998, 102, 4288– 4294. (18) Manne, S.; Gaubt, H. E. Science 1995, 270, 1480–1482. (19) Kaggwa, G. B.; Froebe, S.; Huynh, L.; Ralston, J.; Bremmell, K. Langmuir 2005, 21, 4695–4704. (20) Adler, J. J.; Singh, P. K.; Patist, A.; Rabinovich, Y. I.; Shah, D. O.; Moudgil, B. M. Langmuir 2000, 16, 7255–7262. (21) Arita, T.; Kanda, Y.; Hamabe, H.; Ueno, T.; Watanabe, Y.; Higashitani, K. Langmuir 2003, 19, 6723–6729. (22) Nestor, J.; Esquena, J.; Solans, C.; Luckham, P. F.; Musoke, M.; Levecke, B.; Booten, K.; Tadros, T. F. J. Colloid Interface Sci. 2007, 311, 430–437. (23) Traini, D.; Young, P. M.; Rogueda, P.; Price, R. Pharm. Res. 2007, 24, 125– 135. (24) Traini, D.; Young, P. M.; Rogueda, P.; Price, R. Int. J. Pharm. 2006, 320, 58–63. (25) Raghavan, S. L.; Trividic, A.; Davis, A. F.; Hadgraft, J. Int. J. Pharm. 2001, 212, 213–221. (26) Ziller, K. H.; Rupprecht, H. H. Pharm. Ind. 1990, 52, 1017–1022. (27) Raghavan, S. L.; Schuessel, K.; Davis, A.; Hadgraft, J. Int. J. Pharm. 2003, 261, 153–158.
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(Parsippany, NJ). Hydroxypropyl cellulose (HPC -SSL) was generously gifted by Nippon Soda Co. Ltd. Japan. Methods. Preparation of Drug Substrate. 150 mg of crystalline drug powder was weighed and added to a die designed for making pellets for infrared spectroscopy. The drug in the die was compressed in a Carver press at a pressure of 5 tons for 10 min, after which the pellet was carefully removed from the die. To achieve a smooth drug pellet surface, a piece of mica, with the same diameter as the die, was placed on one of the faces of the die plunger. Pellets were handled using forceps only along their circumference in order to prevent contamination of the top surface of the pellet where polymer adsorption was to be studied. Surface Roughness by AFM. The drug pellet was glued to a microscope glass slide and placed under the AFM microscope. Images were captured in air using a MFP-3D atomic force microscope (Asylum Research, Santa Barbara, CA, USA) in the intermittent-contact mode. Silicon probe cantilevers OMCLAC160TS (Olympus) with nominal spring constants of 42 N/m were used to image surface topography of the initial drug pellets. All measurements were performed at room temperature, and images were analyzed with IgorPro 5.05A software. Preparation of Stabilizer Solutions. 250 mL of the stabilizer solution was prepared by dissolving the required amount of stabilizer in water prepared using a Milli-Q system (Millipore) to obtain the desired concentration. To minimize the potential of drug dissolution from the surface during adsorption studies, the stabilizer solution was saturated with drug. An excess of drug was added to the stabilizer solution, and the suspension was stirred for 24 h at room temperature. Excess of undissolved drug was removed by filtering the suspension through 0.1 μm filters to obtain a saturated solution of the drug. Geometry of Adsorbed Stabilizer by AFM. The drug pellet was immersed vertically in the stabilizer solution (saturated with the drug) to allow for the adsorption of the stabilizer. After 15 min, the pellet was removed from the stabilizer solution and washed with 1 mL of distilled water (saturated with drug). Three such washings were done to remove any excess stabilizer. To obtain a dried pellet, distilled water was blotted off by pressing a tissue paper along the circumference of the pellet. Care was taken not to touch the pellet surface before AFM scanning. The pellet was then dried under nitrogen for 10 min to remove excess moisture from the pellet surface. Intermittent-contact mode technique was employed to obtain AFM images of the adsorbed polymer on the drug substrate. Silicon cantilevers with a nominal spring constant of 2 N/m (Olympus OMCL-AC240TS) were utilized. Images were processed with IgorPro 5.05A software. Such results obtained under ambient conditions are particularly significant as most of the pharmaceutical nanosuspensions often need to be converted into dry powders due to physical or chemical instability of the drug in the aqueous environment for long periods of time during storage. Blank Pellet Preparation. The drug pellet was immersed vertically in the distilled water (saturated with the drug). After 15 min, the pellet was removed from the distilled water and washed with 1 mL of distilled water (saturated with drug). Three such washings were performed, and the sample was handled as described above for visualization of adsorbed polymer. Adhesion Forces. After imaging the adsorbed polymer on the ibuprofen surface, adhesion between the AFM probe and the various polymer-adsorbed surfaces was quantified. The AFM sensitivity was first determined against the ibuprofen surface (with no polymer adsorbed), and then spring constant was calculated via the widely employed Sader method. Regions with and without adsorbed polymer were then marked and numbered. Ten such spots were marked for each region. The probe was finally moved to each of these spots, and force curves were collected. In such force curve measurements, the tip and the sample are initially separated, and thus negligible forces are recorded. Upon contact and indentation, the tip experiences repulsive forces, the slope of Langmuir 2009, 25(21), 12481–12487
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Article Table 1. Particle Size, Solubility, and ζ Potential Data of Ibuprofen Nano/Microsuspensions intensity weighted particle size (nm) following storage at 25 °C
stabilizer
Poloxamer 188 Poloxamer 407 PVP K-30 HPMC K3 HPMC E3 HPMC E5 HPMC E15 HPMC A15
solubility (mg/mL)
ζ potential (mV)
mean
s.d.
mean
s.d.
mean
s.d.
mean
s.d.
mean
s.d.
mean
s.d.
1037 1878 733 921 744 816 846 753
116 130 69 48 62 6 22 18
1133 2110 1130 995 796 854 948 824
104 264 73 8 27 29 47 46
1229 2534 1298 1016 811 911 903 854
70 63 42 49 34 22 49 21
1390 2855 1610 1121 888 873 967 861
34 30 48 101 45 12 14 52
0.060 0.306 0.048 0.058 0.059 0.056 0.064 0.059
0.0005 0.0006 0.0005 0.0001 0.0006 0.0002 0.0003 0.0006
-11.09 -8.79 -13.42 -12.23 -4.83 -4.10 -5.96 -7.34
1.01 2.73 1.06 2.32 2.64 2.22 1.05 1.13
initial
day 1
day 3
day 7
which relates to the substrate stiffness. Most relevant to this work, however, is the attractive force between tip and sample which occurs just before tip-sample separation. This adhesion force should correlate with the efficacy of the various polymers in mitigating agglomeration. A caveat to such measurements is the possible transfer of the polymer from the substrate to the probe apex, causing contamination that could modify subsequent adhesion results. However, this would also degrade image quality and change the cantilever resonant frequency, which is particularly sensitive to mass changes on the probe. Neither effect was observed for any polymers studied.
Results In a previous study, we investigated the ability of various stabilizers (PVP K-30, Poloxamer 188, Poloxamer 407, and various grades of HPMCs (K3, E3, E5, E15, and A15)) for the preparation and stabilization of ibuprofen nanosuspensions. An increase in particle size on storage at 25 °C (Table 1) was observed in formulations made with Poloxamer 188, Poloxamer 407, and PVP. The increase in particle size in the Poloxamer 407 suspensions was attributed to Ostwald ripening due to the high solubility of ibuprofen in the Poloxamer 407 solutions (Table 1). However, the solubility data did not explain the particle size increase observed with PVP and Poloxamer 188 formulations, since ibuprofen exhibited low solubilities in solutions of these stabilizers, which were comparable to that obtained in solutions of the HPMCs. On examination of ζ potential data (Table 1), it was observed that the HPMC formulations exhibited lower negative zeta potential values in comparison to PVP and Poloxamer 188 formulations. It was postulated that specific adsorption behavior of the HPMCs (such as surface coverage, geometry of adsorbed layer, and its strength) may be responsible for masking the inherent negative charge (due to ionization of carboxylic acid group) of the ibuprofen particles to a higher extent than other stabilizers. It was speculated that the HPMC interfacial layer afforded better protection of the ibuprofen nanoparticles against Ostwald ripening or aggregation. Although the solubility and zeta potential data provided some insight into the mechanism of stabilization of ibuprofen nanosuspensions, more substantial evidence was necessary to confirm the above hypothesis. The main objective of this paper is to demonstrate direct evidence of the role of the characteristics of the adsorbed layer on nanosuspension stabilization. Atomic force microscopy was used to elucidate the arrangement/conformation of the stabilizer molecules on the ibuprofen surface. In addition, adhesion forces between the AFM tip and the adsorbed stabilizer were determined to obtain an understanding of the nature of the interactions involved in the adsorption process. Figure 1 shows the height image of the surface of the blank ibuprofen pellet (control experiment). The root-mean-square Langmuir 2009, 25(21), 12481–12487
Figure 1. Height image of bare ibuprofen surface captured in air using intermittent-contact mode.
Figure 2. Height image of HPMC adsorbed on ibuprofen surface captured in air using intermittent-contact mode.
(rms) roughness of the height was 14.0 nm. No features can be observed on the blank ibuprofen pellet surface. Figure 2 illustrates the morphology and arrangement of the HPMC adsorbed on the ibuprofen pellet. The polymeric chains are completely uncoiled and are adsorbed in an open extended conformation on the ibuprofen surface. The height of the chains was approximately 2.7 nm, while the width of the chains is measures 43 nm. This chain width is wider than expected for individual molecules; however, this can be attributed to well-known tip curvature convolution effects when imaging tubular structures with AFM images,28 presuming radially (28) Lee, S. H.; Sigmund, W. M. JOM 2007, 59, 30–33.
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Figure 3. Height (left) and amplitude (right) AFM images of PVP adsorbed on ibuprofen surface captured in air using intermittent-contact mode.
Figure 4. Height (left) and amplitude (right) AFM images of Poloxamer 188 adsorbed on ibuprofen surface captured in air using intermittent-contact mode.
symmetric molecules the height and width are each 2.7 nm across. Figure 3 shows a height image of PVP adsorbed on the ibuprofen surface, with the corresponding amplitude image also included, as it allows better visualization of feature edges. It can be seen that the polymer is adsorbed in globule-like structures with heights ranging from 12 to 28 nm and widths between 100 and 200 nm. In addition, preferential adsorption can be observed on certain faces of the ibuprofen crystal due to crystallographic anisotropy. These structures appear to be swollen single molecules in a coiled conformation or aggregates of coiled molecules of PVP. In addition, these globular structures are predominantly adsorbed on the atomic steps of the ibuprofen crystal. Adsorption of Poloxamer 188 on ibuprofen surface is depicted in Figure 4. Poloxamer 188 adsorbs as slightly elongated structures which are about 1.5 nm in height, 42 nm wide, and 70 nm long. Moreover, similar to PVP preferential adsorption can be detected along the crystal’s atomic steps. Poloxamer 407 adsorption on the ibuprofen surface is shown in Figure 5. Multiple scans of the same area resulted in migration of the adsorbed Poloxamer 407. Similar multiple scans with all other polymers did not reveal any tendency for migration or dislocation of the adsorbed polymer. HPC adsorbed on the ibuprofen surface in an extended open chain pattern similar to HPMC (Figure 6). The observed heights and widths were also comparable to those observed for HPMC approximately 3 and 40 nm, respectively. 12484 DOI: 10.1021/la9016432
Average adhesion forces and 95% confidence values between a single silicon probe and first the bare ibuprofen surface, then the ibuprofen surface with adsorbed polymer, are listed in Table 2. Comparisons within each column are therefore meaningful, while conclusions between columns are difficult to draw, as a new probe was used for each new pair of bare and adsorbed polymer measurements. Accordingly, significant differences in the adhesion forces between the bare surface and the regions with adsorbed polymer are clearly revealed for PVP. A minor difference in for HPMC and the bare ibuprofen surface is apparent, though barely statistically significant. No difference is observed between Poloxamer 188 and the bare ibuprofen surface.
Discussion Atomic force microscopy has been widely used to investigate the adsorption of surfactants and polymers on model surfaces. Materials with extremely smooth surfaces (root-mean-square roughness less than 1 nm) such as freshly cleaved mica and pyrolytic graphite offer ideal surfaces for adsorption studies. Graphite is hydrophobic in nature, whereas untreated mica affords a hydrophilic surface. In addition, mica has been chemically processed in many different ways to obtain surfaces with varying degrees of hydrophobicity. Much knowledge about the adsorption process and the properties of the interfacial layers can be achieved through working with these model systems. However, the surface characteristics of the solid substrates play Langmuir 2009, 25(21), 12481–12487
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Figure 5. Height AFM images of Poloxamer 407 adsorbed on ibuprofen surface captured in air using intermittent-contact mode. First scan (top image), second scan (middle image), and third scan (bottom image) of the same area exhibiting dislocation of the polymer.
an important role in the adsorption process and affect the critical properties of the adsorbed layer.16 Both specific interactions (such as hydrogen bonding) with the exposed surface groups on the adsorbent and nonspecific hydrophobic interactions (between the adsorbent and the adsorbate) can greatly influence the adsorption behavior.17 Therefore, in this study pellets of the drug powder itself were used to mimic the nanoparticle drug suspensions as Langmuir 2009, 25(21), 12481–12487
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closely as possible. One of the key impediments in the use of the actual material of interest is the ability to obtain surfaces with comparable smoothness to mica or graphite. To overcome this limitation, we compressed the ibuprofen crystals in a Carver press using mica on one of the faces of the die plunger to obtain smooth pellets and studied the adsorption behavior of nonionic polymeric stabilizers on it. As shown in Figures 2-5, it is evident that HPMC was the best polymer among those investigated in terms of interaction with the ibuprofen surface to provide an effective surface coverage. HPMC is a substituted cellulosic polymer with methoxy and hydroxypropyl substitution at the 1, 3, or 6 positions of the repeating anhydroglucose units. We hypothesize that interactions between the hydrophobic backbone of HPMC and the hydrophobic groups present on the ibuprofen surface were responsible for its extensive and strong adsorption. High affinity of the HPMC for the ibuprofen surface also causes the molecules to adsorb in an open-chain-like pattern as compared to a compact/ coiled structure. PVP and Poloxamer 188, on the other hand, did not interact well with the ibuprofen surface. The low level of interaction with these polymers is obvious through the shape of the structures formed. Both of these polymers tended to adsorb in more compact/coiled shaped structures, indicating a general lack of affinity for the surface. In addition, in the case of PVP an asymmetric or patchy adsorption of polymer was observed with preferential adsorption on one face of the crystal as compared to the other. This clearly demonstrates why HPMC-based formulations exhibited superior characteristics compared to PVP and Poloxamer 188 based formulations on storage. This confirms our hypothesis that specific superior arrangement of HPMC was responsible for the formation of stable ibuprofen nanosuspensions. Moreover, this provides a much required technique that can assist in stabilizer selection for nanosuspension formulations based on knowledge of the interactions between the drug and the stabilizer. In addition, useful insight into the strength of the interactions between the stabilizer and the drug particle surface were gained through the atomic force microscopy studies. The strength of the interactions between the adsorbate (ibuprofen surface) and adsorbent (stabilizer) plays an important role in the stabilization of the disperse systems. While scanning HPMC, PVP, and Poloxamer 188 samples for adsorption, no migration of the adsorbed stabilizer was observed over multiple scans of the same image. Although identical soft cantilevers were employed and the imaging mode was not changed for the ibuprofen samples with Poloxamer 407, large-scale migration of this poorly adsorbing polymer was observed during imaging. Figure 5 shows the initial and subsequent scans of the same area during imaging of Poloxamer 407. This clearly suggests that Poloxamer 407 had very weak interactions with the ibuprofen surface, and thus, the molecules became easily dislocated as a result of the force applied by the AFM probe. To further test both the hypotheses that adsorbed layer characteristics affect the nanosuspension formation and stability and that AFM is a useful tool for nanosuspension stabilizer selection, several successful nanosuspension formulations were selected from the literature and investigated using AFM. After investigation of a range of stabilizers including Poloxamer 188, Poloxamer 407, PEG, and PVP, Lee et al.7 reported that only HPC formed ibuprofen nanosuspensions. Accordingly, the interaction of HPC with ibuprofen was investigated using AFM (Figure 6). It can be seen that HPC interacts strongly with the surface groups of ibuprofen and adsorbs extensively onto the ibuprofen surface. It adsorbs in an extended open-chain pattern DOI: 10.1021/la9016432
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Figure 6. Height (left) and amplitude (right) AFM images of HPC adsorbed on ibuprofen surface captured in air using intermittent-contact mode. Table 2. Mean Adhesion Force (10 Forces Curves) between Silicon Tip and Either Bare Ibuprofen Surface or Ibuprofen with Adsorbed Polymer A: HPMC bare surface adsorbed polymer
mean (nN) confidence (nN) 10.73 9.05
0.40 1.33
B: PVP
mean (nN) confidence (nN) C: Poloxamer 188 mean (nN) confidence (nN)
bare surface adsorbed polymer
similar to HPMC. However, subtle differences are apparent when compared to HPMC. In the case of HPMC, a more branched pattern was observed, while HPC chains appear to be more linear. This may be attributed to differences in the interaction of the polymers with the ibuprofen surface or to the characteristics of the polymeric chain itself due to variations in the level of hydroxyl group substitution along the cellulosic backbone. The images obtained for polymer adsorption on the ibuprofen pellets revealed the interesting feature that the PVP and the Poloxamer 188 stabilizers tended to adsorb along the lines of atomic steps (Figures 3 and 4). In addition, preferential adsorption is seen on one crystal face in the case of PVP. The X-ray diffraction crystal structure of racemic ibuprofen was obtained from Cambridge Crystallographic Data Center (CCDC), Cambridge, U.K.29,30 The network of molecules was generated by using Accelrys discovery studio using the X-ray crystallographic information. Analysis of the crystal structure of racemic ibuprofen (Figure 7) indicates that the molecules of ibuprofen are arranged as dimers in the crystal lattice. The carboxylic acid groups of the two molecules in the dimer form hydrogen bonds with each other and the molecules are stacked along their aromatic rings. This molecular arrangement leads to a hydrophobic crystal surface with all the hydrogen bonding groups buried deep inside. It is only at the atomic steps or specific crystal faces where these groups are available for interaction with the polymers (as evident in Figure 3). It thus appears that the hydrogen bonding of the CdO group of the PVP molecule with the exposed COOH group at the atomic steps of the ibuprofen crystal surface is responsible for adsorption of PVP. PVP has been previously documented to form hydrogen bonds with the COOH group of the ibuprofen in a number of studies.31 The adsorption pattern observed with Poloxamer 188 can also be attributed to a similar hydrogen bonding pattern. (29) Shankland, N.; Wilson, C. C.; Florence, A. J.; Cox, P. J. Acta Crystallogr., Sect. C: Cryst. Struct. Commun. 1997, 53, 951–954. (30) Cambridge Crystallographic Data Centre, Cambridge, U.K. (31) Bogdanova, S.; Pajeva, I.; Nikolova, P.; Tsakovska, I.; M€uller, B. Pharm. Res. 2005, 22, 806–815.
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12.33 20.50
0.27 2.81
bare surface adsorbed polymer
25.17 25.88
1.05 0.83
Figure 7. Crystal structure of racemic ibuprofen.
These results may explain some of the aspects of the success of the top-down approach toward drug particle preparation (i.e., milling large particles to smaller sizes in the presence of stabilizers) over the bottom-up approach (i.e., precipitating small particles and then stabilizing) for nanosuspension preparation. During milling, particle fracture occurs along all possible directions exposing some of the groups that are usually buried deep inside the crystal. A favorable interaction between the exposed groups and the stabilizer may assist in polymer adsorption and subsequent stabilization of the nanoparticles. Although these carboxylic groups or other interacting groups (depending on the specific drug) may also become exposed during a precipitation process, the rapid particle growth will limit polymer adsorption due to insufficient time for interaction and competing reactions. For example, the time scale for particle growth during the precipitation processes is on the order of a few microseconds, Langmuir 2009, 25(21), 12481–12487
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whereas the typical milling time in a top-down process is on the order of minutes to several hours. Analysis of the adhesion force data reveals that the adhesion force of the AFM probe with the bare ibuprofen surface was 10.73 nN, 12.33 nN, and 25.17 nN for HPMC, PVP, and Poloxamer 188, respectively. This initial variation can be explained by the fact that new tips are used for each experiment and the contact area of the tip with the ibuprofen surface will vary due to minute differences in the radius of each AFM tip. Moreover, the imaging experiments were conducted in air, and environmental humidity may play a role in the observed differences of the initial adhesion force values. However, for each polymer studied the same AFM tip was used to collect data on the bare surface as well as the same surface with adsorbed polymer. Furthermore, measurements on each set of bare and coated surfaces were acquired in the same lab session, during which humidity did not change appreciably. As described previously, the AFM tips exhibited stronger attractive forces with adsorbed PVP molecules as compared to the bare ibuprofen surface. The greater adhesion forces observed on adsorbed PVP regions may be attributed to capillary forces due to the water present in the swollen polymer molecules. For HPMC, the adhesion forces on polymers were less attractive as compared to the bare surface, while no difference in adhesion forces was observed for Poloxamer 188. To summarize this set of measurements, the adhesion forces on modified surfaces compared to the bare surfaces were substantially greater for PVP, equal for Poloxamer 188 and slightly less for HPMC according to 95% confidence error bars. The trend of these differences in the adhesion profiles matches well with the observed stability of corresponding ibuprofen nanosuspensions. Now that this protocol is established, a comprehensive study between ibuprofen surfaces prepared as described above and ibuprofen particles attached to the apex of the standard AFM probes (instead of the silica AFM probe itself) is planned for future research. This will directly quantify the influence of
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Article
stabilizers on interparticulate forces in dried nanosuspensions particles. Similar studies in liquid will also be performed, in order to provide complementary guidelines for aqueous systems.
Conclusions An atomic force microscopy (AFM) method was developed and successfully employed to study the interaction of various nanosuspension stabilizers with a drug surface. The technique is based on uniquely prepared samples of ibuprofen crystals that are atomically flat over micrometer-scale distances. Upon adsorption of various stabilizing polymers onto these surfaces in solution, AFM is then used to visualize the resulting adsorption morphology, affording direct evidence of the suitability of the stabilizers for the production of stable nanosuspensions. The stabilization efficacy of these nonionic polymeric stabilizers was shown to correlate with surface coverage and adhesion. Furthermore, the AFM images revealed subtle differences in the specific adsorption geometry for the polymers, with smooth and regularly branched adhesions performing optimally while clustered polymers did not stabilize the suspensions. This may aid in the future selection of suitable excipients for the preparation of nanosuspensions. It appears that the success of the top-down processing method over bottom-up approaches is due to the constant generation of new surfaces, which continuously expose specific chemical groups that can interact with the stabilizer, thus enhancing interaction and stabilization. In addition, depending upon the specific application, this method can be applicable to the selection of excipients for other unit operations such as granulation. Acknowledgment. We gratefully acknowledge the financial support from Dane.O.Kildsig Center of Pharmaceutical Processing and Research. Dr. Bryan Huey would also like to recognize the support from NSF-ENGR-CMMI-Nanobiomechanics award 0626231.
DOI: 10.1021/la9016432
12487
