8/13/2019 Scanning Probe Microscopy Method for Nanosuspension Stabilizer Selection

1/7

DOI:10.1021/la9016432 12481Langmuir 2009,25(21),1248112487 Published on Web 09/30/2009

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 interactionof polymeric stabilizers with ibuprofen to determine their suitability for the preparation and stabilization of ibuprofennanosuspensions. Images obtained clearly showed that HPMC and HPC interacted strongly with the ibuprofenresulting in extensive surface adsorption, confirming their suitability for ibuprofen nanosuspension preparation. Inaddition, differences in the morphology of the adsorbed HPMC and HPC molecules were observed, which may beattributed to their variable degree of substitution. Consistent with their poor performance in stabilizing the ibuprofennanosuspensions,imagesobtained with PVPand Poloxamers 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 top-down processing for the preparation of nanosuspensions as compared to bottom-up processing. On the basis of therelationship 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 scientificrationale for nanosuspension stabilizer selection rather than the trial and error method which is currently practiced.

Introduction

Nanosuspensions are defined as colloidal dispersions of dis-crete drug particles with particle size in the range 1-1000 nm.They are being actively pursued for the formulation of pharma-ceutical compounds with poor aqueous solubility. The small sizeand high surface area of drug nanosuspensions result in increaseddissolution rates and hence improved bioavailability of waterinsoluble compounds and thus can overcome the basic hurdle ofpoor dissolution rate in the successful development of thesecompounds. Besides improved bioavailability,1,2 altered disposi-tion,3 increased chemical stability,4 increased drug loading,5 andreduced toxicity and side effects6 are additional biopharmaceu-tical advantages of the nanosuspensions.

Any nanosuspension formulation has three basic ingredients:active pharmaceutical ingredient (API), stabilizer, and dispersionmedium. Usually, the dispersion medium is water, and the API is ahydrophobic drug compound with poor aqueous solubility. Thestabilizers are surface active agents or polymers that adsorb at theinterface of the drug particles with water. Stabilizerscommonlyusedto stabilize nanosuspensions include polymers (such as polyvinylpyrrolidone (PVP), hydroxypropyl methyl cellulose (HPMCs), andhydroxypropyl cellulose (HPCs)), ionic surfactants (e.g., sodiumdodecyl sulfate (SDS)), and nonionic surfactants (e.g., Tweens andPoloxamers (polyoxyethylene-polyoxypropylene copolymers)).Ionic surfactants stabilize suspensions via electrostatic repulsion,while polymers and nonionic surfactants facilitate suspension stabi-lity via steric repulsion.

The high surface area of drug nanoparticles, by the virtue ofwhichthey exhibit their unique biopharmaceutical characteristics,also renders them thermodynamically unstable5 and promotesagglomeration and crystal growth. This high surface area in-creases the total free energy (G = s/l* A) of the system, whereA is thetotal surfaceareaof theparticles ands/l is the interfacialtension between the drug surface and aqueous phase. Conse-quently, stabilizers are an indispensible component of nanosus-pension formulations, since they adsorb at the interface, reducingthe interfacial tension (s/l) and thereby decreasing the total free

energy G (G = s/l* A) of the system. Stabilizers playa crucial role in the formation, stability, and overall performanceof nanosuspensions.7,8 Both nanoparticle creation and subse-quent stabilization are highly sensitive to the choice of stabilizer.Paradoxically, the current practice used to select stabilizers is atrial and error approach with no prior knowledge of their abilityto interact with the drug surface.9,10

Recently, a few studies focused on developing empirical rela-tionships between stabilizer efficacy and some property of thedrug compound have been published to aid in stabilizer selection.In one such study, Choi et al.11 investigated the stabilizingefficiency of polymers as a function of similarity between thesurface energies of the polymer and the drug. They concludedthat, though comparing surface energies may provide some help,

specific interactions between the stabilizer and the drug appearedto play a more important role in the formation of the nanosus-pensions. Lee et al.7 prepared nanosuspensions of several waterinsoluble drugs with various polymers in an attempt to establish ageneral guide for the preparation of nanosuspensions. A rule of

*Corresponding author. Diane J. Burgess. Phone: 860-486-3760. Fax: 860-486-0538. E-mail: d.burgess@uconn.edu.

(1) Liversidge, G. G.; Cundy, K. C. Int. J. Pharm.1995,125, 9197.(2) Kraft, W. K.; Steiger, B.; Beussink, D.; Quiring, J. N.; Fitzgerald, N.;

Greenberg, H. E.; Waldman, S. A.J. Clin. Pharmacol. 2004, 44, 6772.(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, 615619.

(5) Rabinow, B. E.Nat. Rev. Drug Discovery 2004,3, 785796.(6) Liversidge, G. G.; Conzentino, P.Int. J. Pharm.1995,125, 309313.

(7) Lee, J.; Choi, J. Y.; Park, C. H. Int. J. Pharm.2008,355, 328336.(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, 20912103.

(9) Jacobs, C.; Muller, R. H.Pharm. Res.2002,19, 189194.(10) Lee, J.; Lee, S. J.; Choi, J. Y.; Yoo, J. Y.; Ahn, C. H.Eur. J. Pharm. Sci.

2005,24, 441449.(11) Choi, J. Y.; Yoo, J. Y.; Kwak, H. S.; Nam, B. U.; Lee, J. Curr. Appl. Phys.

2005,5, 472474.

8/13/2019 Scanning Probe Microscopy Method for Nanosuspension Stabilizer Selection

2/7

12482 DOI: 10.1021/la9016432 Langmuir2009,25(21),1248112487

Article Verma et al.

thumb was proposed that drugs with high molecular weights, lowsolubility, high melting points, and surface energies similar tothat of the stabilizer used could be successfully processed intonanosuspensions. Similarly, Eerdenbrugh et al.8 investigated theformation of nanosuspensions of several drugs with variouspolymeric stabilizers. In this study, it was concluded that thehydrophobicity of the drug played a significant role in theproduction of the drug nanoparticles. However, none of thesestudies 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 wasto investigate local interactions between the stabilizer and thedrug surface to determine whether this may assist formulationscientists in the judicious choice of stabilizers. Atomic forcemicroscopy (AFM) was employed for the first time to investigatedrug-stabilizer interactions through visualization of stabilizersadsorbed directlyonto thedrug surface. Although AFM has beenused successfully in thepast forthe imaging of soft structures suchas adsorbed surfactant and polymersonto solid substrates in bothgaseous and liquid environments, all measurements were per-formed with substrates easily acquired with a nearly atomicallyflat surface (e.g., mica, silica, or graphite).12-18 Direct visualiza-

tion of the adsorbed stabilizer directly on the drug surface willtherefore not only aid in comprehending the extent of interactionbetween the drug and the stabilizer, but also afford vital informa-tion about the characteristics of the adsorbed layer such as thespecific arrangement of the stabilizer molecules, the thicknessof the adsorbed layer, and the strength of the interfacial film.All of these characteristics are known to significantly affectwettability,19 interparticulate interactions,20-24 crystal growth,Ostwald ripening, and aggregation.25-27 A comprehensiveknowl-edge on specific drug-stabilizer interactions and other funda-mental details about the exact nature of the interfacial adsorbedlayer will thus pave the way toward a more scientific basis forstabilizers 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 gradewas generously gifted by Dow Chemical Company (Midland,MI). Poloxamer 188 (Pluronic F-68), Poloxamer 407 (PluronicF-127), and Kollidon 30 (PVP K-30) were purchased from BASF

(Parsippany, NJ). Hydroxypropyl cellulose (HPC -SSL) wasgenerously 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 designedfor making pellets for infrared spectroscopy. The drug in the diewas compressed in a Carver press at a pressure of 5 tons for10 min, after which the pellet was carefully removed from the die.To achieve a smooth drug pellet surface, a piece of m ica, with thesame diameter as the die, was placed on one of the faces of the dieplunger. Pellets were handled using forceps only along theircircumference in order to prevent contamination of the top sur-face of the pellet where polymer adsorption was to be studied.

Surface Roughness by AFM. The drug pellet was glued toa microscope glass slide and placed under the AFM micro-scope. Images were captured in air using a MFP-3D atomic forcemicroscope (Asylum Research, Santa Barbara, CA, USA) in theintermittent-contact mode. Silicon probe cantilevers OMCL-AC160TS (Olympus) with nominal spring constants of 42 N/mwere used to image surface topography of the initial drug pellets.All measurements were performed at room temperature, andimages were analyzed withIgorPro 5.05Asoftware.

Preparation of Stabilizer Solutions.250 mL of the stabilizersolution was prepared by dissolving the required amount ofstabilizer in water prepared using a Milli-Q system (Millipore)to obtain the desired concentration. To minimize the potential ofdrug dissolution from the surface during adsorption studies, thestabilizer solution was saturated with drug. An excess of drug wasadded to the stabilizer solution, and the suspension was stirred for24 h at room temperature. Excess of undissolved drug wasremoved by filtering the suspension through 0.1 m filters toobtain a saturated solution of the drug.

Geometry of Adsorbed Stabilizer by AFM. The drug pelletwas immersed vertically in the stabilizer solution (saturated withthe drug) to allow for the adsorption of the stabilizer. After15 min, the pellet was removed from the stabilizer solution andwashed with 1 m L of distilled water (saturated with drug). Threesuch washings were done to remove any excess stabilizer. Toobtain a dried pellet, distilled water was blotted off by pressinga tissue paper along the circumference of the pellet. Care was

taken not to touch the pellet surface before AFM scanning. Thepellet was then dried under nitrogen for 10 min to remove excessmoisture from the pellet surface. Intermittent-contact modetechnique was employed to obtain AFM images of the adsorbedpolymer on the drug substrate. Silicon cantilevers with a nominalspring constant of 2 N/m (Olympus OMCL-AC240TS) wereutilized. Images were processed with IgorPro 5.05A software.Such results obtained under ambient conditions are particularlysignificant as most of the pharmaceutical nanosuspensions oftenneed to be converted into dry powders due to physicalor chemicalinstabilityof the drugin the aqueous environment forlong periodsof time during storage.

Blank Pellet Preparation. The drug pellet was immersedvertically in the distilled water (saturated with the drug). After15 min, the pellet was removed from the distilled water and

washed with 1 m L of distilled water (saturated with drug). Threesuch washings were performed, and the sample was handled asdescribed above for visualization of adsorbed polymer.

Adhesion Forces.After imaging the adsorbed polymer on theibuprofen surface, adhesion between the AFM probe and thevarious polymer-adsorbed surfaces was quantified. The AFMsensitivitywas first determined against the ibuprofen surface (withno polymer adsorbed), and then spring constant was calculatedvia the widely employed Sader method. Regions with and withoutadsorbed polymer were then marked and numbered. Ten suchspots were marked for each region. The probe was finally movedto each of these spots, and force curves were collected. In suchforce curve measurements, the tip and the sample are initiallyseparated, and thus negligible forces are recorded. Upon contactand indentation, the tip experiences repulsive forces, the slope of

(12) Arita, T.; Kanda, Y.; Higashitani, K. J. Colloid Interface Sci. 2004, 273,102105.

(13) Ducker, W. A.; Grant, L. M.J. Phys. Chem.1996, 100, 1150711511.(14) Fleming, B. D.; Wanless, E. J. Microsc. Microanal.2000,6, 104112.(15) Fleming, B. D.; Wanless, E. J.; Biggs, S. Langmuir1999, 15, 87198725.(16) Grant, L. M.; Ducker, W. A.J. Phys. Chem. B 1997,101, 53375345.(17) Grant, L. M.; Tiberg, F.; Ducker, W. A. J. Phys. Chem. B1998,102, 4288

4294.

(18) Manne, S.; Gaubt, H. E.Science1995,270, 1480

1482.(19) Kaggwa, G. B.; Froebe, S.; Huynh, L.; Ralston, J.; Bremmell, K.Langmuir

2005, 21, 46954704.(20) Adler, J. J.; Singh, P. K.; Patist, A.; Rabinovich, Y. I.; Shah, D. O.;

Moudgil, B. M. Langmuir2000,16, 72557262.(21) Arita, T.; Kanda, Y.; Hamabe, H.; Ueno, T.; Watanabe, Y.; Higashitani,

K.Langmuir2003,19, 67236729.(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, 430437.(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. 2006,320,5863.

(25) Raghavan,S. L.;Trividic,A.; Davis,A. F.;Hadgraft, J. Int.J. Pharm. 2001,212, 213221.

(26) Ziller, K. H.; Rupprecht, H. H. Pharm. Ind.1990,52, 10171022.(27) Raghavan, S. L.; Schuessel, K.; Davis, A.; Hadgraft, J.Int. J. Pharm.2003,

261, 153158.

8/13/2019 Scanning Probe Microscopy Method for Nanosuspension Stabilizer Selection

3/7

DOI: 10.1021/la9016432 12483Langmuir 2009,25(21),1248112487

Verma et al. Article

whichrelates to the substrate stiffness. Most relevantto this work,however, is the attractive force between tip and sample whichoccurs just before tip-sample separation. This adhesion forceshould correlate with the efficacy of the various polymers inmitigating agglomeration. A caveat to such measurements is thepossible transfer of the polymer from the substrate to the probeapex, causing contamination that could modify subsequent adhe-sion results. However, this would also degrade image quality andchange the cantilever resonant frequency, which is particularlysensitive to mass changes on the probe. Neither effect was

observed for any polymers studied.

Results

In a previous study, we investigated the ability of variousstabilizers (PVP K-30, Poloxamer 188, Poloxamer 407, andvarious grades of HPMCs (K3, E3, E5, E15, and A15)) for thepreparation and stabilization of ibuprofen nanosuspensions. Anincrease in particle size on storage at 25 C (Table1) was observedin formulations made with Poloxamer 188, Poloxamer 407, andPVP. The increase in particle size in the Poloxamer 407 suspen-sions wasattributed to Ostwald ripening due to thehigh solubilityof ibuprofen in the Poloxamer 407 solutions (Table 1).

However, the solubility data did not explain the particle sizeincrease observed with PVP and Poloxamer 188 formulations,

since ibuprofen exhibited low solubilities in solutions of thesestabilizers, which were comparable to that obtained in solutionsof the HPMCs. On examination of potential data (Table 1),it was observed that the HPMC formulations exhibited lowernegative zeta potential values in comparison to PVP and Polo-xamer 188 formulations. It was postulated that specific adsorp-tion behavior of the HPMCs (such as surface coverage, geometryof adsorbed layer, and its strength) may be responsible formasking the inherent negative charge (due to ionization ofcarboxylic acid group) of the ibuprofen particles to a higherextent than other stabilizers. It was speculated that the HPMCinterfacial layer afforded better protection of the ibuprofennanoparticles against Ostwald ripening or aggregation. Althoughthe solubility and zeta potential data provided some insight intothe mechanism of stabilization of ibuprofen nanosuspensions,more substantial evidence was necessary to confirm the abovehypothesis.

The main objective of this paper is to demonstrate directevidence of the role of the characteristics of the adsorbed layeron nanosuspension stabilization. Atomic force microscopy wasused to elucidate the arrangement/conformation of the stabilizermolecules on the ibuprofen surface. In addition, adhesion forcesbetween theAFM tip andthe adsorbed stabilizer were determinedto obtain an understanding of the nature of the interactionsinvolved in the adsorption process.

Figure 1 shows the height image of the surface of the blankibuprofen pellet (control experiment). The root-mean-square

(rms) roughness of the height was 14.0 nm. No features can beobserved on the blank ibuprofen pellet surface. Figure 2 illustratesthe morphology and arrangement of the HPMC adsorbed on theibuprofen pellet. The polymeric chains are completely uncoiled andare adsorbed in an open extended conformation on the ibuprofensurface. The height of the chains was approximately 2.7 nm, whilethe width of the chains is measures 43 nm. This chain width is widerthan expected for individual molecules; however, this can beattributed to well-known tip curvature convolution effects whenimaging tubular structures with AFM images,28 presuming radially

Table 1. Particle Size, Solubility, and Potential Data of Ibuprofen Nano/Microsuspensions

intensity weighted particle size (nm) following storage at 25 C

stabilizer initial day 1 day 3 day 7 solubility (mg/mL) potential (mV)

mean s.d. mean s.d. mean s.d. mean s.d. mean s.d. mean s.d.

Poloxamer 188 1037 116 1133 104 1229 70 1390 34 0.060 0.0005 -11.09 1.01Poloxamer 407 1878 130 2110 264 2534 63 2855 30 0.306 0.0006 -8.79 2.73PVP K-30 733 69 1130 73 1298 42 1610 48 0.048 0.0005 -13.42 1.06HPMC K3 921 48 995 8 1016 49 1121 101 0.058 0.0001 -12.23 2.32

HPMC E3 744 62 796 27 811 34 888 45 0.059 0.0006 -

4.83 2.64HPMC E5 816 6 854 29 911 22 873 12 0.056 0.0002 -4.10 2.22HPMC E15 846 22 948 47 903 49 967 14 0.064 0.0003 -5.96 1.05HPMC A15 753 18 824 46 854 21 861 52 0.059 0.0006 -7.34 1.13

Figure 1. Height image of bare ibuprofen surface captured in airusing intermittent-contact mode.

Figure 2. Height image of HPMC adsorbed on ibuprofen surfacecaptured in air using intermittent-contact mode.

(28) Lee, S. H.; Sigmund, W. M.JOM2007,59, 3033.

8/13/2019 Scanning Probe Microscopy Method for Nanosuspension Stabilizer Selection

4/7

12484 DOI: 10.1021/la9016432 Langmuir2009,25(21),1248112487

Article Verma et al.

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 thepolymer 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 ofPoloxamer 188 on ibuprofen surface is depicted in Figure 4.

Poloxamer 188 adsorbs as slightly elongated structures which areabout 1.5 nm in height, 42 nm wide, and 70 nm long. Moreover,

similar to PVP preferential adsorption can be detected along the

crystals atomic steps.Poloxamer407 adsorption on theibuprofen 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 ordislocation of the adsorbed polymer. HPC adsorbed on the

ibuprofen surface in an extended open chain pattern similar toHPMC (Figure 6). The observed heights and widths were also

comparable to those observed for HPMC approximately 3 and

40 nm, respectively.

Average adhesion forces and 95% confidence values between asingle silicon probe and first the bare ibuprofen surface, then theibuprofen surface with adsorbed polymer, are listed in Table 2.Comparisons within each column are therefore meaningful, whileconclusionsbetween columns aredifficult to draw, as a new probewas used for each new pair of bare and adsorbed polymermeasurements. Accordingly, significant differences in the adhe-sion forces between the bare surface and the regions withadsorbed polymer are clearly revealed for PVP. A minor differ-ence in for HPMC and the bare ibuprofen surface is apparent,though barely statistically significant. No difference is observedbetween Poloxamer 188 and the bare ibuprofen surface.

Discussion

Atomic force microscopy has been widely used to investigatethe adsorption of surfactants and polymers on model surfaces.Materials with extremely smooth surfaces (root-mean-squareroughness less than 1 nm) such as freshly cleaved mica andpyrolytic graphite offer ideal surfaces for adsorption studies.Graphite is hydrophobic in nature, whereas untreated micaaffords a hydrophilic surface. In addition, mica has been chemi-cally processed in many different ways to obtain surfaceswith varying degrees of hydrophobicity. Much knowledge aboutthe adsorption process and the properties of the interfacial layerscan be achieved through working with these model systems.However, the surface characteristics of the solid substrates play

Figure 3. Height (left) and amplitude (right) AFM images of PVP adsorbed on ibuprofen surface captured in air using intermittent-contactmode.

Figure 4. Height (left) and amplitude (right) AFM images of Poloxamer 188 adsorbed on ibuprofen surface captured in air usingintermittent-contact mode.

8/13/2019 Scanning Probe Microscopy Method for Nanosuspension Stabilizer Selection

5/7

DOI: 10.1021/la9016432 12485Langmuir 2009,25(21),1248112487

Verma et al. Article

an important role in the adsorption process and affect the criticalproperties of the adsorbed layer.16 Both specificinteractions (suchas hydrogen bonding) with the exposed surface groups on theadsorbent and nonspecific hydrophobic interactions (between theadsorbent and the adsorbate) can greatly influence the adsorptionbehavior.17 Therefore, in this study pellets of the drug powderitself were used to mimic the nanoparticle drug suspensions as

closely as possible. One of the key impediments in the use of theactual material of interest is the ability to obtain surfaces withcomparable smoothness to mica or graphite. To overcome thislimitation, we compressed the ibuprofen crystals in a Carver pressusing micaon one of thefacesof thedie plunger to obtain smoothpellets and studied the adsorptionbehavior of nonionic polymericstabilizers on it.

As shown in Figures 2-5, it is evident that HPMCwas the bestpolymer 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 andhydroxypropyl substitution at the 1, 3, or 6 positions of therepeating anhydroglucose units. We hypothesize that interactionsbetween the hydrophobic backbone of HPMC and the hydro-phobic groups present on the ibuprofen surface were responsiblefor its extensive and strong adsorption. High affinity of theHPMC for the ibuprofen surface also causes the molecules toadsorb in an open-chain-like pattern as compared to a compact/coiled structure. PVP and Poloxamer 188, on the other hand, didnot interact well with the ibuprofen surface. The low level ofinteraction with these polymers is obvious through the shape ofthe structures formed. Both of these polymers tended to adsorb inmore compact/coiled shaped structures, indicating a general lack

of affinity for the surface. In addition, in the case of PVP anasymmetric or patchy adsorption of polymer was observed withpreferential adsorption on one face of the crystal as compared tothe other. This clearly demonstrates why HPMC-based formula-tions exhibited superior characteristics compared to PVP andPoloxamer 188 based formulations on storage. This confirms ourhypothesis that specific superior arrangement of HPMC wasresponsible for the formation of stable ibuprofen nanosuspen-sions. Moreover, this provides a much required techniquethat canassist in stabilizer selection for nanosuspension formulationsbased on knowledge of the interactions between the drug andthe stabilizer.

In addition, useful insight into the strength of the interactionsbetween the stabilizer and the drug particle surface were gained

through the atomic force microscopy studies. The strength of theinteractions between the adsorbate (ibuprofen surface) and ad-sorbent (stabilizer) plays an important role in the stabilizationof the disperse systems. While scanning HPMC, PVP, andPoloxamer 188 samples for adsorption, no migration of theadsorbed stabilizer was observed over multiple scans of the sameimage. Although identical soft cantilevers were employed and theimaging mode was not changed for the ibuprofen samples withPoloxamer 407, large-scale migration of this poorly adsorbingpolymer was observed during imaging. Figure 5 shows the initialand subsequent scans of the same area during imaging ofPoloxamer 407. This clearly suggests that Poloxamer 407 hadvery weak interactions with the ibuprofen surface, and thus, themolecules became easily dislocated as a result of the force appliedby the AFM probe.

To further test both the hypotheses that adsorbed layercharacteristics affect the nanosuspension formation and stabilityand that AFM is a useful tool for nanosuspension stabilizerselection, several successful nanosuspension formulations wereselected from the literature and investigated using AFM. Afterinvestigation of a range of stabilizers including Poloxamer 188,Poloxamer 407, PEG, and PVP, Lee et al.7 reported that onlyHPC formed ibuprofen nanosuspensions. Accordingly, the inter-action of HPC with ibuprofen was investigated using AFM(Figure 6). It can be seen that HPC interacts strongly with thesurface groups of ibuprofen and adsorbs extensively onto theibuprofen surface. It adsorbs in an extended open-chain pattern

Figure 5. Height AFM images of Poloxamer 407 adsorbed onibuprofen 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 thepolymer.

8/13/2019 Scanning Probe Microscopy Method for Nanosuspension Stabilizer Selection

6/7

12486 DOI: 10.1021/la9016432 Langmuir2009,25(21),1248112487

Article Verma et al.

similar to HPMC. However, subtle differences are apparent whencompared to HPMC. In the case of HPMC, a more branchedpattern wasobserved, while HPC chainsappear to be morelinear.This may be attributed to differences in the interaction of thepolymers with theibuprofen surface or to thecharacteristicsof thepolymeric chain itself due to variations in the level of hydroxylgroup substitution along the cellulosic backbone.

The images obtained for polymer adsorption on the ibuprofenpellets revealed the interesting feature that the PVP and thePoloxamer 188 stabilizers tended to adsorb along the linesof atomic steps (Figures 3 and 4). In addition, preferentialadsorption is seen on one crystal face in the case of PVP. The

X-ray diffraction crystal structure of racemic ibuprofen wasobtained from Cambridge Crystallographic Data Center(CCDC), Cambridge, U.K.29,30 The network of molecules wasgenerated by using Accelrys discovery studio using the X-raycrystallographic information. Analysis of the crystal structure ofracemic ibuprofen (Figure 7) indicates that the molecules ofibuprofen are arranged as dimers in the crystal lattice.

The carboxylic acid groups of the two molecules in the dimerform hydrogen bonds with each other and the molecules arestacked along their aromatic rings. This molecular arrange-ment leads to a hydrophobic crystal surface with all thehydrogen bonding groups buried deep inside. It is only at theatomic steps or specific crystal faces where these groups areavailable for interaction with the polymers (as evident in

Figure 3). It thus appears that the hydrogen bonding of theCdO group of the PVP molecule with the exposed COOHgroup at the atomic steps of the ibuprofen crystal surface isresponsible for adsorption of PVP. PVP has been previouslydocumented to form hydrogen bonds with the COOH group ofthe ibuprofen in a number of studies.31 The adsorption patternobserved with Poloxamer 188 can alsobe attributed to a similarhydrogen bonding pattern.

These results may explain some of the aspects of the successof the top-down approach toward drug particle preparation(i.e., milling large particles to smaller sizes in the presence ofstabilizers) over the bottom-up approach (i.e., precipitating smallparticles and then stabilizing) for nanosuspension preparation.During milling, particle fracture occurs along all possible direc-tions exposing some of the groups that are usually buried deepinside the crystal. A favorable interaction between the exposedgroups and the stabilizer may assist in polymer adsorption andsubsequent stabilization of the nanoparticles. Although thesecarboxylic groups or other interacting groups (depending onthespecificdrug)may also become exposed duringa precipitationprocess, the rapid particle growth will limit polymer adsorptiondue to insufficient time for interaction and competing reactions.For example, the time scale for particle growth during theprecipitation processes is on the order of a few microseconds,

Figure 6. Height (left) and amplitude (right) AFM images of HPC adsorbed on ibuprofen surface captured in air using intermittent-contactmode.

Table 2. Mean Adhesion Force (10 Forces Curves)between Silicon Tip and Either Bare Ibuprofen Surface or Ibuprofen with Adsorbed Polymer

A: HPMC mean (nN) confidence (nN) B: PVP mean (nN) confidence (nN) C: Poloxamer 188 mean (nN) confidence (nN)

bare surface 10.73 0.40 bare surface 12.33 0.27 bare surface 25.17 1.05

adsorbed polymer 9.05 1.33 adsorbed polymer 20.50 2.81 adsorbed polymer 25.88 0.83

Figure 7. Crystal structure of racemic ibuprofen.

(29) Shankland, N.; Wilson, C. C.; Florence, A. J.; Cox, P. J. Acta Crystallogr.,Sect. C: Cryst. Struct. Commun. 1997,53, 951954.

(30) Cambridge Crystallographic Data Centre, Cambridge, U.K.(31) Bogdanova, S.; Pajeva, I.; Nikolova, P.; Tsakovska, I.; M uller, B. Pharm.

Res.2005,22, 806815.

8/13/2019 Scanning Probe Microscopy Method for Nanosuspension Stabilizer Selection

7/7

DOI: 10.1021/la9016432 12487Langmuir 2009,25(21),1248112487

Verma et al. Article

whereas the typical milling time in a top-down process is on theorder of minutes to several hours.

Analysis of the adhesion force data reveals that the adhesionforce of the AFM probe with the bare ibuprofen surface was10.73 nN, 12.33 nN, and 25.17 nN for HPMC, PVP, andPoloxamer 188, respectively. This initial variation can be explainedby the fact that new tips are used for each experiment and thecontact area of the tip with the ibuprofen surface will vary due tominute differences in the radius of each AFM tip. Moreover, the

imaging experiments were conducted in air, and environmentalhumidity may play a role in the observed differences of the initialadhesion force values. However, for each polymer studied the sameAFM tip was used to collect data on the bare surface as well as thesame surface with adsorbed polymer. Furthermore, measurementson each set of bare and coated surfaces were acquired in the samelab session, during which humidity did not change appreciably.

As described previously, the AFM tips exhibited strongerattractive forces with adsorbed PVP molecules as compared tothe bare ibuprofen surface. The greater adhesion forces observedon adsorbed PVP regions may be attributed to capillary forcesdue to the water present in the swollen polymer molecules. ForHPMC, the adhesion forces on polymers were less attractive ascompared to the bare surface, while no difference in adhesion

forces was observed for Poloxamer 188. To summarize this setof measurements, the adhesion forces on modified surfacescompared to the bare surfaces were substantially greater forPVP, equal for Poloxamer 188 and slightly less for HPMCaccording to 95% confidence error bars. The trend of thesedifferences in the adhesion profilesmatches wellwith the observedstability of corresponding ibuprofen nanosuspensions.

Now that this protocol is established, a comprehensive studybetween ibuprofen surfaces prepared as described above andibuprofen particles attached to the apex of the standard AFMprobes (instead of the silica AFM probe itself) is planned forfuture research. This will directly quantify the influence of

stabilizers on interparticulate forces in dried nanosuspensionsparticles. Similar studies in liquid will also be performed, in orderto provide complementary guidelines for aqueous systems.

Conclusions

An atomic force microscopy (AFM) method was developedand successfully employed to study the interaction of variousnanosuspension stabilizers with a drug surface. The technique is

based on uniquely prepared samples of ibuprofencrystals that areatomically flat over micrometer-scale distances. Upon adsorptionof various stabilizing polymers onto these surfaces in solution,AFM is then used to visualize the resulting adsorption morpho-logy, affording direct evidence of the suitability of the stabilizersfor the production of stable nanosuspensions. The stabilizationefficacy of these nonionic polymeric stabilizers was shown tocorrelate with surface coverage and adhesion. Furthermore, theAFM images revealed subtle differences in the specific adsorptiongeometry for the polymers, with smooth and regularly branchedadhesions performing optimally while clustered polymers did notstabilize the suspensions. This may aid in the future selection ofsuitable excipients for the preparation of nanosuspensions. Itappears that the success of the top-down processing method over

bottom-up approaches is due to the constant generation of newsurfaces, which continuously expose specific chemical groups thatcan interact with the stabilizer, thus enhancing interaction andstabilization. In addition, depending upon the specific applica-tion, this method can be applicable to the selection of excipientsfor other unit operations such as granulation.

Acknowledgment. We gratefully acknowledge the financialsupport from Dane.O.Kildsig Center of Pharmaceutical Proces-sing and Research. Dr. Bryan Huey would also like to recognizethe support from NSF-ENGR-CMMI-Nanobiomechanicsaward 0626231.