Polymer-Dispersed Bicontinuous Cubic Glycolipid Nanoparticles

Thomas Abraham,*,† Masakatsu Hato,†,§ and Mitsuhiro Hirai‡

Bionanomaterial and Surface Interactions Group, Nanotechnology Research Institute, AIST, Tsukuba Central 5,Tsukuba 305-8565, Japan, and Department of Physics, Gunma University, 4-2 Aramaki,Maebashi 371-8510, Japan

We found that certain amphiphilic polymers such as PEO-PPO-PEO triblockcopolymer (PL) can directly disperse a cubic glycolipid, 1-O-phytanyl-â-D-xyloside (â-XP), into bicontinuous cubic nanoparticles in water medium. The use of synchrotronsmall-angle X-ray diffraction (SSAXD) permitted the identification of the exactstructure of these dispersed particles in the colloidal state. Dynamic light scatteringmethod was used to obtain particle size distributions. The dispersion quality and thedispersion time can be improved by co-dissolving the lipid and the polymer in a commonsolvent. The mean volume diameter of these dispersed colloidal particles depends onthe mixing time and polymer concentration. About 5 wt % (0.18 mol %) of polymer tolipid weight was found to be sufficient to produce stable colloidal dispersions. At thispolymer content and at 3 h of stirring time, the mean volume diameter of cubic colloidalparticles was found to be 1.0 µm. Increase of dispersion time to 6 h reduced the colloidalparticle size from 1.0 µm to 660 nm. At 3 h of mixing time, the increase of polymercontent, from ∼5 to ∼10 wt %, reduced the particle mean diameter from 1.0 µm to675 nm. Irrespective of these dispersion times and polymer contents, the dispersedcolloidal particles exhibit predominately the Pn3m cubic phase structure, the sameas that of a â-XP-water binary mixture, although a weak coexistence of Im3m cubicphase is identified in these colloidal particles. This coexistence is found to have thecharacteristics of a Bonnet relation, which forms convincing evidence for the infiniteperiodic minimal surface descriptions (IPMS). Considering the biotechnologicalsignificance, the preparation of these colloidal dispersions was carried out in aphosphate-buffered saline (PBS) system. These cubic colloidal dispersions exhibitedgood stability and the cubic phase structure remained intact in the PBS system.

Introduction

To date glyceryl monooleate (GMO) has been the onlylipid of choice in formulating bicontinuous cubic phasedispersions for biotechnology applications (1). Recently,a glycoside, 1-O-phytanyl-â-D-xyloside, which exhibitscubic phase structure, has been reported in the literature(2). Lipids that exhibit bicontinuous cubic phases areimportant because of their unique role in membraneprotein crystallization (3, 4), their relationship to struc-tures seen in biological membranes (5-7)m and theirpotentials as drug carriers (8, 9).

Cubic lipid-water binary systems form reversed bi-continuous cubic phases that are thermodynamicallystable in excess water. The lipid bilayers in cubic phase-water binary systems form a three-dimensional networkthat separates two identical water channels that have awater pore diameter in nanometers in the fully hydratedcubic phase (5). The cubic phases have a three-dimen-sional periodic bicontinuous conduit structure with a high

specific lipid/water interfacial area. Two alternate struc-tural representations have been proposed to describe thebicontinuous cubic phases, one in terms of rodlike ele-ments and the other of folded surfaces (infinite periodicminimal surfaces, IPMS), corresponding to Schoen’sskeletal graphs and infinite labyrinths respectively (10,11). Lately, the structural classifications based on theIPMS are most commonly used to describe cubic phaselipid structures. Very recently, representations in termsof nodal surfaces have been introduced to describe thedynamic structure of cubic phases (12). Three differentinverse bicontinuous cubic lipid phases are known atpresent. They are associated with the space groups Pn3m,Ia3d, and Im3m and have corresponding IPMS types ofdiamond (D-surface), gyroid (G-type), and primitive (P-surface), respectively (13, 14).

The possibility to enhance the enzymatic stability ofincorporated drugs and proteins and the ability toincorporate and slowly release a variety of drugs withdifferent physicochemical properties have made the cubicphase an interesting choice for use in the drug deliverysystems (8, 9). The use of cubic bulk phases in drugadministration is, however, limited by their high viscos-ity, which makes them difficult to inject. Therefore,submicron-sized colloidal dispersions of these cubic struc-tures have been proposed as drug delivery systems (15).Compared to liposomes, the high bilayer area to particle

* To whom correspondence should be addressed. Current ad-dress: Department of Biochemistry, 3-39 Medical Sciences Build-ings, University of Alberta, Edmonton, Canada T6G 2H7. Tel:(780) 492-2412. Fax: (780) 492-0886. Email: tabraham@ualberta.ca.

† Bionanomaterial and Surface Interactions Group.‡ Gunma University.§ Current address: Nanotechnology Research Institute, AIST,

Tsukuba Central 5, Tsukuba 305-8565, Japan.

255Biotechnol. Prog. 2005, 21, 255−262

10.1021/bp0498544 CCC: $30.25 © 2005 American Chemical Society and American Institute of Chemical EngineersPublished on Web 11/19/2004

volume ratio of the cubic lipid particles may also increasethe relative payload of hydrophobic drugs.

It has been known for some time that colloidal disper-sions could be prepared from the cubic monoolein-waterphases (15). Several other good descriptions of thepreparation of aqueous dispersions of cubic monoolein-water phases have been provided later (16-18). All ofthese investigations, however, are limited to monooleindispersions. The main objective of this work is to producea new cubic particle dispersion. In this article, we reportthe production of cubic particle colloidal dispersions froma glycoside, 1-O-phytanyl-â-D-xyloside (â-XP), which ex-hibits Pn3m cubic phase in aqueous medium, and thestructural characteristics of these submicron/nano par-ticles. It is interesting to mention that phytanyl lipidchains form a common basic core structure of major lipidsof archaebacterial plasma membranes (28). Archaebac-teria are organisms that grow in exceptional ecologicalcircumstances such as high or low pH, high temperature,and high ionic strength (29). The method of preparationof the cubic colloidal dispersion employed here requiresno significant energy input and offers the advantage ofdirect inclusion of colloidal stabilizers, sensitive drugmaterials, and proteins. An amphiphilic triblock copoly-mer was used to provide dispersion stability. An impor-tant concern here is whether the nanostructured lipid/water bicontinuous cubic networks are preserved in thesubmicron/nano particles. To answer this importantquestion, the structure of these colloidal particles wasinvestigated using synchrotron X-ray diffraction.

Materials and MethodsMaterials. The glycoside 1-O-phytanyl-â-D-xyloside (â-

XP), which exhibits cubic Pn3m phase in excess aqueousmedium at low temperatures, was used (Figure 1, a). Thesynthesis and the relevant physical properties of â-XPare described elsewhere (2, 19, 20). Phosphate-bufferedsystem (PBS) was purchased from Sigma-Aldrich, Japan.Pluronic F127 (PL), a triblock copolymer with the com-position of [EO]99-[PO]67-[EO]99 was obtained from BASF,USA (Figure 1b). All cubic colloidal dispersions wereprepared in ELGA purified water of resistivity 18.2 MΩ‚cm.

Synchrotron-Small-AngleX-rayDiffraction(SSAXD).The phase structures of dispersed â-XP colloidal particleswere determined using a synchrotron-small-angle X-raydiffraction spectrometer (SSAXD), attached to a synchro-tron radiation source (High Energy Accelerator ResearchOrganization (KEK), Tsukuba, Japan). The â-XP disper-sions of concentrations ca. 4-6% w/w were directly usedin SAXD measurements. Sample solutions were filledinto a mica-window cell with 1.0-mm path length. A smallsuction pump was used for the filling. The cell was placedin a cell holder controlled at 25 ( 0.1 °C. The X-raywavelength used was 0.149 nm. A camera length of 80cm was used. The exposure time was 480 s (8 min) for

each sample. A one-dimensional position-sensitive pro-portional counter was used to detect the scatteringintensities. The counter consisted of 512 channels andwas calibrated using collagen. The scattering data wasdirectly transferred to a computer, where the scatteringprofile (intensity vs channel number) was displayed.

In a diffraction experiment the Bragg peaks of cubicstructure are observed at

where dhkl is the Bragg spacing, a is the unit cell size,and h, k, and l are the Miller indices. The Miller indicesdepend on the lattice type and the symmetry elementsof the cubic structure. In well-ordered cubic structures,the Pn3m lattice exhibits characteristics reflections(peaks) at (1, 1, 0), (1, 1, 1), (2, 0, 0), (1, 1, 0), (2, 1, 1), (2,2, 0), (2, 2, 1), (3, 1, 0), (3, 1, 1), (2, 2, 2), and (3, 2, 1),whereas the peak positions of Im3m occur at (1, 1, 0), (2,0,0), (2, 1, 1), (2, 2, 0), (3, 1, 0), (3, 1, 1), (2, 2, 2), (3, 2, 1),(4, 0, 0), (4, 1, 1), (3, 3, 0), and (4, 2, 0). In the diffractionplots, the scattering vector q () 4π sin θ/λ, where λ isthe wavelength of X-ray beam) versus the intensity isplotted.

Small-Angle X-ray Diffraction (SAXD). In the caseof the bulk â-XP-water binary mixture, SAXD measure-ments were performed with Ni-filtered Cu KR radiation(wavelength ) 0.154 nm) generated by a Rigaku RU-200X-ray generator (40 kV, 100 mA) with a double pinholecollimator (0.5 mm φ - 0.3 mm φ).

A syringe assembly was used to prepare the bulkmixture of â-XP with water. This mechanical mixingdevice consisted of two microsyringes with threadedterminations (250 µL, ITO Microsyringe: MS-GAN025)and a stainless steel coupler with a small threaded boreand a Teflon O-ring at the center of the bore. The syringeswere connected by the coupler through which the samplecomponents were subjected to pass during the mixingprocess. This assembly ensures thorough mixing andeliminates the possibility of loss of water or change incompositions. Samples of total weight ca. 100 mg wereused. Appropriate quantities of â-XP and water wereweighed into the syringe. The sample loaded syringe wasthen kept in a chamber saturated with water vapor atroom temperature for about 3 days for incubation. Toensure the homogeneity of the system, the sample wassubjected to thorough mixing repeatedly during incuba-tion. The binary mixture, prepared as above, was directlytransferred into a quartz capillary for SAXD measure-ments. The sample-loaded capillaries were then incu-bated at 25 °C for at least 2 days. The SAXD measure-ments were carried out at 25 ( 0.1 °C. The samples wereequilibrated at this temperature for 1-2 h prior to X-rayexposure. The sample temperature was controlled witha Mettler FP82HT hot stage within an accuracy of (0.1°C). The sample to film distance was 205 mm. Theexposure time was 60 min. To ensure the homogeneityof the system, three to four sample-loaded capillarieswere prepared from the same system.

Polarized Microscopy. A polarized microscope (PM)was used to inspect the optical texture of lipid liquidcrystalline samples. This is a simple method to distin-guish the isotropic mesophase such as the cubic phasesfrom the anisotropic lamellar phase. The liquid crystal-line phases formed during the preparation and the finaldispersion products were investigated optically usingpolarized microscopy.

Figure 1. Chemical structures of (a) 1-O-phytanyl-â-D-xyloside(â-XP) and (b) triblock copolymer, Pluronic F127 (PL).

dhkl ) a

xh2 + l2 + k2(1)

256 Biotechnol. Prog., 2005, Vol. 21, No. 1

Dynamic Light Scattering Method. The particlesize distribution of the cubic particle dispersions wasdetermined using UPA 150 particle size analyzer. Thisinstrument is capable of measuring particle sizes rangingfrom 3.0 nm to 7.0 µm. Concentrated dispersions werediluted to 1.0 wt % in order to adjust the signal level.All measurements were performed at 25 °C. In a typicalexperiment the dispersions of about 5.0 mL were directlypoured into the sample cell. The experimental run timewas about 10.0 min. The UPA uses the controlledreference method of dynamic light scattering. In prin-ciple, the particle size distribution is computed directlyfrom measured frequency spectrum recovered from theDoppler-shifted scattered light, assuming the particlegeometry is spherical or nonspherical. In this case, aspherical geometry was assumed. The data inputs for thecomputation include the particle refractive index (1.37)and particle density (0.97 g/cc). The particle size, themean volume diameter, is defined as

where ni, vi, and di are number, volume, and diameter ofthe ith species

Results and AnalysisPreparation of Cubic Lipid Colloidal Disper-

sions. The aqueous solubility of â-XP is in the order of10-6 M or less. The â-XP-water binary mixtures formbicontinuous cubic phases. At water contents above 20wt %, a cubic Pn3m phase exists in equilibrium with anaqueous phase at 25 °C (2). In a typical dispersionexperiment, first the â-XP was incubated overnight withwater at 60:40 lipid to water weight ratio. After overnightincubation, simple additions of 5.1 wt % polymer (PL) tolipid weight and water (to obtain a ∼5 wt % â-XPdispersion) and subsequent stirring in a glass vesselproduced milky white cubic dispersion. The dispersionprocess was followed using a polarized microscope. After10 min of continuous stirring, lipid fragments of a widerange of sizes ranging from 100 µm to 1.0 mm appearedand the system turned milky white. As the stirringprocess continued, lipid particles of submicron sizes wereformed and the dispersion became more milky white. InFigure 2, we show the particle size distribution ofcolloidal lipid dispersion obtained after 3 h of continuousstirring (broken lines). Particles of size ranging from 200nm to 6.0 µm are present, and the mean volume diameterof the particle is determined as 1.4 µm. The volumefraction of particles larger than 1.0 µm is about 66% ofthe total volume of particles. No birefringence regionsare seen in these dispersed particles under the polarizedmicroscope, suggesting that these mixtures are isotropic.Note that the isotropic behavior is a typical characteristicof the cubic phase.

The colloidal dispersion thus produced was examineddirectly using SSAXD (curve A in Figure 3). At leastseven well-defined peaks (reflections) of varying intensi-ties are seen, as indicated by broken lines. The indexingof diffraction pattern confirms cubic Pn3m phase and unitcell size (a) of 9.55 nm (Table 1). The diffractogram of afully hydrated â-XP-water (32.4/67.6) bulk binary sys-tem generated by SAXD is also shown in Figure 3 (curveC). The diffraction peaks of the bulk binary mixture(curve C), unlike those of colloidal dispersions, appearbroader because this particular diffractogram was gener-ated using conventional SAXD. Note that the colloidal

dispersions were examined using synchrotron-SAXD,which is capable of providing sharper peaks as a resultof its high-intensity X-ray beams (see Materials andMethods for details). It can be noted that the diffractionpeaks of the cubic colloidal particles are indistinguishablefrom those of the Pn3m structure of a fully hydratedâ-XP-water (32.4/67.6) bulk binary system, but with amarginally higher unit cell size. This bicontinuous cubicliquid crystalline phase (Pn3m), as others, is a structur-ally twisted lamellar phase and contains a single bilayermembrane (note that ideal lamellar liquid crystallinephase consists of disjointed parallel bilayers). The centerof the bilayer, whose locus is the minimal surface, andthe interconnecting congruent water channels reside inthe apolar region and in the polar region, respectively.The bilayer can be considered as folded in a spaceaccording to an infinite periodic minimal surface, theD-surface, with zero mean curvature (7).

Additional weak reflections were also seen, as indicatedby downward arrows in Figure 3. Seemingly, the colloidaldispersion exhibits more than one form of bicontinuouscubic phase structures. A plausible explanation is thatit may result from the formation of additional new cubic

mean volume diameter ) ∑nividi

∑nivi

(2)

Figure 2. (a) Particle size distributions in â-XP colloidaldispersions. (- - -) Colloidal dispersion, 5.7 wt % â-XP dispersioncontaining 5.1 wt % PL to lipid weight, prepared from the freshlymixed system; particle sizes ranging from 0.2 to 6.0 µm arepresent. (s) Colloidal dispersion prepared from the premixedlipid-polymer mixture, 4.8 wt % â-XP dispersion containing 5.7wt % PL to lipid weight; particle sizes ranging from 0.2 to 2.8µm are seen. See text for details.

Figure 3. Synchrotron-SAXD and SAXD diffraction profiles.(A) Colloidal dispersion, 5.7 wt % â-XP dispersion containing5.1 wt % PL to lipid weight, produced from freshly mixed lipid-polymer system. (B) Colloidal dispersion, 4.8 wt % â-XP disper-sion containing 5.7 wt % PL to lipid weight, produced from thepremixed lipid-polymer system. (C) Fully hydrated pure â-XP-water (32.4 wt % â-XP) binary system. The diffraction data aresummarized in Table 1. Broken lines denote the cubic Pn3mdiffraction peaks; downward arrows indicate cubic Im3m peaks.See text for details.

Biotechnol. Prog., 2005, Vol. 21, No. 1 257

phase structures. An attempt has been made to assignthe extra peak positions to other bicontinuous cubicsymmetries. Indexing of these extra peak positionsconfirms a cubic Im3m phase with a unit cell size (a) of12.79 nm. According to an IPMS description, in thisparticular cubic phase the center of the bilayer mimicsthe P-surface (7). It appears that two cubic phases, Pn3mand Im3m, could coexist in these colloidal particles. Also,it is interesting to mention that both phases observedgive a diffraction peak at d ) 3.02 nm (see Table 1, boldentries), corresponding to the same interplanar distanceand thus relating the [310] of the Pn3m to the [411(330)]of the Im3m phases, i.e., d310(Pn3m) ) d411(330)(Im3m).This observation suggests that epitaxially related cubicphases, Pn3m and Im3m, might exist in this system. Notethat epitaxial relationship means the relationships oforientation and geometry between the two adjacentphases (14).

In an effort to increase the dispersion quality in termsof particle size and particle size distribution, the lipidand the polymer were premixed in a common solvent. Itwas found that acetone dissolves both the â-XP and PL.In a typical experiment (see Figure 4), the â-XP and 5.7wt % PL to lipid weight were dissolved in acetone.Thereafter acetone was removed by slow evaporationunder reduced pressure (vacuum). The resulting mixtureof the lipid and the polymer was further dried undervacuum to remove small traces of acetone. The â-XP-PL binary mixture thus prepared was incubated withwater overnight at lipid/water weight ratio 60/40. Afterovernight incubation, an appropriate quantity of waterwas added to the system to produce a 4.8 wt % â-XPdispersion. The system was then subjected to stirring ina glass vessel using a magnetic stirrer. The systemturned into a milky white dispersion after 10 min ofstirring. Compared to the freshly mixed system, in thispremixed system most of the lipid fragments are smallat this stage. The particle size distribution of thesecolloidal particles produced after 3 h of stirring time isshown in Figure 2. The particle size distribution isdistinctly different from those obtained for the freshlymixed system. The colloidal particles produced from thepremixed system show homogeneous particle size andsize distribution. Particle sizes ranging from 200 nm to2.8 µm are present, and the mean particle diameter isfound to be 1.0 µm. The volume fraction of particles largerthan 1.0 µm is only about 47% of the total volume ofparticles. Clearly, the premixing of â-XP and PL providesbetter dispersion quality in terms of particle size and sizedistribution. These dispersed colloidal particles exhibitthe bicontinuous cubic phase structure (curve B in Figure

3), and the other structural aspects remain the same asthat of the colloidal particles produced from the freshlymixed system.

Table 1. Diffraction Data (Figure 3)

colloidal dispersionsa bulk â-XP-water mixtureb

Pn3m Im3m Pn3m

hkl d (nm) a (nm) hkl d (nm) a (nm) hkl d (nm) a (nm)

110 6.76 9.55 110 9.04 12.79 110 6.53 9.23111 5.54 9.59 200 nsc 111 5.32 9.22200 4.80 9.59 211 5.15 12.62 200 4.57 9.14211 3.89 9.53 220 nsc 211 3.72 9.11220 3.36 9.50 310 nsc 220 3.23 9.12221 3.17 9.52 222 3.71 12.84 221 3.05 9.14310 3.02 9.55 321 nsc 222 2.64 9.14

400 nsc

411(330) 3.02 12.82420 2.88 12.89

av ) 9.55 av ) 12.79 av ) 9.16a Freshly mixed system (5.7 wt % â-XP dispersion at 5.1 wt % PL) and premixed system (4.8 wt % â-XP dispersion with 5.7 wt % PL)

(curves A and B in Figure 3). b 32.4 wt % â-XP (curve C in Figure 3). c Not shown.

Figure 4. Steps involved in the preparation of colloidal cubicparticles from the premixed â-XP/ PL mixture. See text fordetails.

258 Biotechnol. Prog., 2005, Vol. 21, No. 1

Having shown that the premixing method produceshomogeneous particles size and size distributions, it isinteresting to see further the effects of stirring time andpolymer content on the characteristics of dispersedcolloidal particle size and the structure produced in thesame way. In the following sections we address theseissues in regard to the particle size distribution and thestructure.

Effects of Stirring Time on Colloidal Particle Sizeand Structure. To determine the effects of stirring timeon colloidal particle size distribution and structure, thecolloidal dispersions were prepared at different stirringtimes from the premixed lipid/polymer system. In thesedispersions, the â-XP concentration was ∼5 wt % with∼5 wt % polymer to lipid weight. In Figure 5, we comparethe particle size distributions of dispersions prepared atdifferent stirring times. Increasing the stirring timeappears to reduce the particle size and shift the particlesize distribution to the lower side. As noted in theprevious section, 3 h of stirring time produced colloidalparticles ranging from 200 nm to 2.8 µm. In this casethe mean particle diameter is found to be 1.0 µm andthe fraction of particles in the submicron level is justbelow 50%. After 6 h of stirring time, the system containscolloidal particles ranging from 100 nm to 1.9 µm. Themean volume diameter of colloidal particles in this caseis determined as 650 nm, which is about two-thirds ofthat obtained after 3 h of stirring time. In addition,interestingly the amount of colloidal particles larger than1.0 µm is reduced dramatically from 47% to 14% whenthe stirring time increased from 3 to 6 h. Thus, increasingstirring time from 3 to 6 h appears to have influence onthe particle size distribution. Further increase of thestirring time to 12 h did not appear to make any changein the particle size distribution. The particle size distri-bution remains essentially the same as that of 6 h evenafter 12 h of stirring (Figure 5). On the structural side,irrespective of the stirring time and their particle sizedistributions, all of these colloidal particle dispersionsexhibit similar cubic phase structural features, the sameas that of the colloidal dispersions produced at 3 h ofstirring time.

Effects of Polymer Content on Colloidal ParticleSize and Structure. In Figure 6, we compare theparticle size distributions of 4.2 wt % â-XP dispersionsobtained after 3 h of stirring times, containing 5.7 and10.0 wt % PL to lipid weight. Increase of the polymer

content appears to reduce the particle size and shift theparticle size distribution to the lower side. When thepolymer content increased from 5.7 to 10.0 wt %, themean volume diameter of the colloidal particles reducedfrom 1.0 µm to 670 nm and the fraction of particles largerthan 1.0 µm reduced from 47% to 17%. On the structuralside, the increase of polymer content from 5.7 to 10.0 wt% did not bring any significant changes in the cubicstructural features of these dispersed colloidal particles(Figure 7 and Table 2).

Dispersion Stability. The stability of these disper-sions toward flocculation and aggregation was checkedqualitatively by visual inspection and by using thepolarized microscope (400 times magnification level).These dispersions, ∼5% â-XP, containing ∼5 and ∼10 wt% PL, are stable for at least several months at roomtemperature. In particular, a ∼5% â-XP dispersion, with10 wt % polymer, shows good dispersion stability over 6months. These dispersions exhibited slight creamingphenomena, i.e., a tendency of colloidal particles toflocculate into multiple aggregates, after 2 weeks ofstorage at room temperature. This creaming tendencyincreases with increasing â-XP concentration, whereasit decreases with increasing polymer concentration. Theredispersion of the creamy layer, however, was readilyachieved by manual shaking of the dispersion. Thisimplies that the creaming phenomena (flocculation) oc-curred at the shallow secondary minimum position in theinterparticle potential rather than in the deep primaryminimum where the colloidal particles could be heldtightly by van der Waals attractions. In this case, thepresence of the adsorbed polymer on colloidal particle inthe form of a steric layer could increase the potentialbarrier between primary and secondary minima consid-erably.

The stability of these cubic particle dispersions in saltsolutions is a notable characteristic. These dispersionsare found to be stable even in 300 mM 1:1 monvalent(NaCl) and 150 mM 1:2 divalent (MgCl2) salt solutions,signifying again the role of added triblock polymer (PL)as a steric stabilizer. Note that the steric layers such asthe poly(ethylene oxide) layers (PEO), as in this case, areinsensitive to the added salt conditions (21). Since thesteric repulsive force (due to the presence of stericpolymer layers) originates purely from the excludedmonomer volume interactions, this form of force isunaffected by the added salts. Thus, these dispersion

Figure 5. Effects of stirring time on particle size distributionof cubic dispersions. All the colloidal dispersions, produced fromthe premixed polymer-lipid system, containing ∼5.0 wt % â-XPand ∼5 wt % polymer (PL) to lipid weight: (- - -) after 3 h ofstirring time, particle sizes ranging from 0.2 to 2.8 µm are seen;(s) after 6 h of stirring time, particle sizes ranging from 0.12to 1.8 µm are seen; (‚ ‚ ‚) after 12 h of stirring time. See text fordetails.

Figure 6. Effects of polymer content and PBS on particle sizedistribution of cubic dispersions. All of the colloidal dispersionswere produced from the premixed polymer-lipid system andthe stirring time was 3 h. (- - -) Colloidal dispersion producedat 4.8 wt % â-XP and 5.7 wt % PL to lipid weight. (s) Colloidaldispersion produced at 4.2 wt % â-XP and 10.0 wt % PL to lipidweight. (‚ ‚ ‚) Colloidal dispersion produced at 4.6 wt % â-XPand 10.1 wt % PL to lipid weight in PBS. See text for details.

Biotechnol. Prog., 2005, Vol. 21, No. 1 259

systems exhibit good stability and can be consideredstable in salt solutions. This has significance in manybiotechnological and pharmaceutical applications. Forinstance, in the incorporation process of proteins in thesecubic particles, one has to deal with buffers and back-ground salt solutions. Another interesting area to bementioned is the drug delivery applications, where in vivosystems the stability of drug carriers in salt solutions isan important criterion for such applications.

Cubic Colloidal Dispersions in Phosphate-Buff-ered Saline (PBS). One of the intended applications ofthese cubic colloidal particles is drug delivery systems.With this application in mind, the preparations werecarried out in PBS. Note that PBS means 0.01 Mphosphate-buffered saline (0.138 M NaCl, 0.0027 M KCl,pH 7.4, at 25 °C). In the PBS system, the particle sizedistribution remains the same as that of water (dottedlines in Figure 6). Most importantly, this colloidal particledispersion, prepared in PBS, exhibits similar cubic phasestructural features as that of prepared in water (curveC in Figure 7). Interestingly, the PBS does not changeeither particle size or the bicontinuous cubic structureof these particles, thus opening up future possibilities ofthese particles in drug delivery applications.

DiscussionThe results in general highlight the following. A

triblock amphiphilic copolymer could disperse a bicon-

tinuous cubic glycolipid into submicron/nano particles bya simple stirring method and kinetically stabilize themthereafter by steric mechanism. Premixing the lipid andthe polymer in a common solvent and subsequent incu-bation of the premixed polymer-lipid system in waterovernight appear to improve the dispersion quality. Thecolloidal particle size and the dispersion stability dependon the stirring time and the polymer concentration. About5 wt % of polymer to lipid weight is found to be sufficientto produce stable colloidal dispersions. These dispersedcolloidal particles, stabilized even at ∼10 wt % polymerto lipid, exhibit cubic structural features, thus implyingthat the process or the polymer dosage at this level doesnot induce any significant structural changes. Thesecubic dispersed particles exhibit good stability in saltsolutions and in PBS.

There are few reports describing the preparations andthe structures of polymer-stabilized cubic phase aqueousdispersions based on the cubic phase lipid monoolein (15-18). Gustafsson et al. (16) reported that the dispersedparticles, stabilized with 7.4 wt % PL to lipid, exhibitedIm3m cubic phase. This structure was different from thatof the monoolien-water binary mixtures in excess water.Note that monoolien-water binary mixtures in excesswater exhibit Pn3m cubic phase. This phase transition,induced by the addition of polymer, was in agreementwith the phase diagram of monoolien-water-PL systemsreported earlier by Landh (22). Gustafsson et al. (16)suggested the penetration of the polymer into the lipidlayer as a possible reason for such a structural change.Contrary to these observations, we did not see anysignificant structural changes in our system (the polymer-stabilized cubic particles) induced by polymer even at 10wt % PL to lipid weight. At these polymer concentrations,the colloidal particles exhibited predominantly the Pn3mcubic structure, the same as that of the â-XP-waterbinary system, although a weak coexistence of Im3mcubic phase is evident in these colloidal particles. Inaddition, the dispersed cubic particles show well-definedPn3m reflections. This is most likely due to the structuraldifferences between the GMO and the â-XP. Note thatGMO is an unsaturated lipid that exhibits less packingorder compared to a saturated lipid such as â-XP.Therefore, the penetration of polymer segments in thebilayer hydrophobic regions seems to be much easier inmonoolien compared to that occurs in â-XP.

The cubic particle dispersions exhibit good stabilityover several months, despite the fact that, even at ∼10wt % polymer to lipid weight, the interaction betweenthe bilayer and the polymer segments is not sufficientlylarge to induce any significant structural changes in thePn3m cubic phase. Various microdispersions, includingliposomes, are proved to be stabilized by the amphiphilictriblock copolymer PL (24). In the case of liposomes, theexperimental evidence such as the increase of the hy-drodynamic radii and the reduction of ú potential areindications of some level of incorporation of polymersegments in the bilayer (25). The stabilizing effectobserved in our system emerges possibly from the physi-cal interaction between PPO blocks and the nonpolarregion of cubic assembly. The hydrophobic PPO (polypro-pylene oxide) segment, being in a poor solvent like water,is expected to have the collapsed conformation. The linearchain size of the PPO blocks, i.e., R ) bN1/3 (where b isthe size of the repeating unit and N is the number ofrepeating units), is estimated as 1.0 nm. Conversely, thehydrophilic block PEO, being in a good solvent (water),has the excluded volume conformation according to theFlory radius (R ) bN0.6). The chain size in this case is

Figure 7. Synchrotron-SAXD profiles showing the effects ofpolymer (PL) content and PBS on the structure of dispersedcolloidal particles. All of the colloidal dispersions were producedfrom the premixed polymer-lipid system and the stirring timewas 3 h. (A) Colloidal dispersion produced at 4.8 wt % â-XP and5.7 wt % PL to lipid weight. (B) Colloidal dispersion producedat 4.2 wt % â-XP and 10.0 wt % PL to lipid weight. (C) Colloidaldispersion produced at 4.6 wt % â-XP and 10.1 wt % PL to lipidweight in PBS. See text for details.

Table 2. Diffraction Data (Figure 7)

colloidal dispersion premixed systema

Pn3m Im3m

hkl d (nm) a (nm) hkl d (nm) a (nm)

110 6.98 9.87 110 9.31 13.16111 5.66 9.80 200 nsb

200 4.91 9.82 211 5.28 12.93211 3.99 9.77 220 nsb

220 3.46 9.79 310 nsb

221 3.25 9.74 222 3.71 12.84222 3.08 9.74 321 nsb

400 nsb

411(330) 3.08 13.07420 2.90 12.95

av ) 9.79 av ) 12.79a 4.2 wt % â-XP dispersion with 10 wt % PL to lipid wt (curve

B in Figure 7). b Not shown.

260 Biotechnol. Prog., 2005, Vol. 21, No. 1

estimated as 3.9 nm. Consequently, the linear dimensionof triblock polymer PL is 8.9 nm, which is comparable tothe hydrodynamic size of PL adsorbed onto perflurocar-bon droplets (26). The thickness of lipid bilayer, i.e., thetwice of the thickness of phytanyl chains, is determinedas 2.6 nm (27). The bilayer region of the dispersedparticles can very well accommodate the hydrophobicPPO block. Thus the polymer chains can be consideredto be anchored on the hydrophobic region of the dispersedparticles through the hydrophobic blocks (PPO blocks)of PL. The hydrophilic PEO blocks, on the other hand,would dangle in water that offers steric stability to thecubic particles, thus preventing the possibility of colloidalaggregation.

The coexistence of Pn3m (D-surface) and Im3m (P-surface) cubic phases in the colloidal particles is aninteresting aspect that needs to be emphasized (seeFigure 3 and Table 1). Such coexistence has beenunambiguously identified recently in similar cubic par-ticles produced by a method based on the dialysisprinciple (30). According to the infinite periodic minimalsurface (IPMS) descriptions, D-type and P-type minimalsurfaces correspond to Pn3m and Im3m cubic phases,respectively (7). There is a mathematical relationship(mapping) between the P-type and D-type surfaces,defined by the so-called Bonnet transformation (7). TheBonnet relation means that these surfaces (P and D) canbe transformed into each other by bending with constantGaussian curvature. The Gaussian curvature at all thepoints on the surfaces remains the same after thetransformation, although the surfaces may appear to bequite different after a mathematical mapping. Accordingto the minimal surface theories, when the P and D cubicphases are in coexistence, the ratio between the latticeparameters (a) of the two cubic phases should be 1.28(23). This metric relation arises because the differentialgeometries of P and D minimal surfaces are identical andare simply related by the Bonnet relation (or by math-ematical mapping). The metric relation in our case,between the lattice parameters of coexisting Im3m (P-surface) and Pn3m (D-surface) cubic phases, is deter-mined as

This is close to the theoretical value of 1.28, signifyingthe Bonnet relation between these two phases. Thus theexperimentally determined lattice parameter ratio hasthe characteristics of a Bonnet relation, which providesa compelling case for the existence of cubic phases withminimal surface (IPMS) structures in these colloidalparticles. Note that this phase transformation, which isdriven by entropy, does not involve an enthalpy change(∆H ) 0). The corresponding phases, Pn3m and Im3m,can thus be closely related from an energetic point ofview. The relative geometry and the energy of these twocubic phases are fixed as long as the monolayer interfaceis parallel to the underlying minimal surfaces.

ConclusionsAn amphiphilic triblock polymer such as PEO-PPO-

PEO triblock copolymer (PL) was found to disperse acubic glycolipid, 1-O-phytanyl-â-D-xyloside (â-XP), intobicontinuous cubic nanoparticles in water medium. Thesynchrotron-SAXD measurements unambiguously estab-lished the bicontinuous cubic phase structure of thedispersed colloidal particles. The mean volume diameter

of these dispersed colloidal particles depends on themixing time and the polymer concentration. About 5 wt% of polymer to lipid weight was found to be sufficientto produce stable colloidal dispersions of bicontinuouscubic phase having dimension of 660 nm. Irrespective ofthe dispersion time and the polymer content, the bicon-tinuous cubic phase structure of the dispersed colloidalparticles was found to be predominantly Pn3m phase, thesame as that of a â-XP-water binary mixture. TheBonnet relation that has been identified in the structuraldetermination provided convincing evidence for the in-finite minimal surface (IPMS) description of the cubicphase structures. These dispersed bicontinuous cubicparticles exhibit good stability in salt solutions and inphosphate-buffered saline, signifying their impendingapplications in biotechnological and pharmaceutical ar-eas.

AcknowledgmentT.A. thanks the Japan Society for Promotion of Science

for the JSPS award and for funding of this research workthrough the JSPS fellowship program. The authors aregrateful to Drs. D. Kato, D. Negishi, and Y. Abe at LionCorporation, Process Development Research Center, forproviding the UPA 150 Particle Size Analyzer facility forthe particle size measurements. Synchrotron-SAXD wasperformed under the approval of the Photon FactoryProgram Advisory Committee (proposal nos. 2001G359and 2003G137).

References and Notes(1) Larsson, K. Aqueous dispersions of cubic lipid-water phases.

Curr. Opin. Colloid Interface Sci. 2000, 5, 64-69.(2) Hato, M.; Minamikawa, H.; Salkar, R. A.; Matsutani, S.

Alkylglycosides with an isoprenoid-type hydrophobic chaincan afford greater control of aqueous phase structures at lowtemperatures. Langmuir 2002, 18, 3425-3429.

(3) Landau, E. M.; Rosenbusch, J. P. A novel concept for thecrystallization of membrane proteins. Proc. Natl. Acad. Sci.U.S.A. 1996, 93, 14532-14535.

(4) De Kruijff, B. Lipids beyond the bilayer. Nature 1997, 386,129-130.

(5) Larsson, K. Cubic lipid-water phases: structures andbiomembrane aspects. J. Phys. Chem. 1989, 93, 7304-7314.

(6) Landh, T. Cubic cell membrane architectures. Thesis, LundUniversity; Sweden, 1996.

(7) Hyde, S.; Andersson, A.; Larsson, K.; Blum, Z.; Landh, T.;Lidin, S.; Ninham, B. W. The Language of Shape. The Roleof Curvature in Condensed Matter: Physics, Chemistry andBiology. Elsevier: New York, 1997.

(8) Drummond, C. J.; Fong, C. Surfactant self-assembly objectsas novel drug delivery vehicles. Curr. Opin. Colloid interfaceSci. 2000, 4, 449-456.

(9) Shah, J. C.; Sadhale, Y.; Chilukuri, D. M. Cubic phase gelsas drug delivery systems. Adv. Drug Delivery Rev. 2001, 47,229-250.

(10) Luzzati, V.; Delacroix, H.; Gulik, A.; Gulik-Krzywicki, T.;Mariani, P.; Vargas, R. Curr. Top. Membr. 1997, 44, 3-24.

(11) Schoen, A. H. Infinite periodic minimal surfaces withoutself-intersections. NASA Technical Note 1970, D-5541.

(12) Jacob, M.; Andersson, S. Finite periodicity and crystalstructure. Z. Kristallogr. 1997, 212, 486-492.

(13) Templer, R. H. Thermodynamic and theoretical aspects ofcubic mesophases in nature and biological amphiphiles. Curr.Opin. Colloid Interface Sci. 1998, 3, 255-263.

(14) Seddon, J. M. Structure of the inverted hexagonal (HII)phase, and nonlamellar phase transitions of lipids. Biochim.Biophys. Acta 1990, 1031, 1-69.

(15) Landh, T.; Larsson, K. Particles, methods of preparing saidparticles and uses thereof. U.S. Patent 5,531,925, 1996.

(16) Gustafsson, J. H.; Wahren, H. L.; Almgren, M.; Larsson,K. Submicron particles of reversed lipid phases in water

aIm3m

aPn3m) 12.79 nm

9.55 nm) 1.33 (2)

Biotechnol. Prog., 2005, Vol. 21, No. 1 261

stabilized by a nonionic amphiphilc polymer. Langmuir 1997,13, 6964-6971.

(17) Spicer, P. T.; Hayden, K. L.; Lynch, M. L.; Ofori-Boateng,A.; Burns, J. L. Novel process for producing cubic crystallinenanoparticles (cubosomes). Langmuir 2001, 17, 5748-5756.

(18) Nakano, M.; Sugita, A.; Matsuoka, H.; Handa, T. Small-angle X-ray scattering and 13C NMR investigation on theinternal structure of cubosomes. Langmuir 2001, 17, 3917-3922.

(19) Minamikawa, H.; Murakami, T.; Hato, M. Synthesis of 1,3-di-O-alkyl-2-O-(â-glycosyl) glycerols bearing oligosaccharidesas hydrophilic groups. Chem. Phys. Lipids 1994, 72, 111-118.

(20) Minamikawa, H.; Hato, M. Phase behavior of syntheticphytanyl-chained glycolipid/water systems. Langmuir 1997,13, 2564-2571.

(21) Kuhl, T. L.; Leckband, D. E.; Lasic, D. D.; Israelachvili, J.N. Modulation of interaction forces between bilayers exposingshort-chained ethylene oxide headgroups. Biophys. J. 1994,66, 1479.

(22) Landh, T. Phase behavior in the system pine oil monoglyc-erides-poloxamer 407-water at 20 °C. J. Phys. Chem. 1994,98, 8453-8467.

(23) Hyde, S. T. Identification of lyotropic liquid crystallinemesophases. In Handbook of Applied Surface and ColloidChemistry; Holmberg, K., Ed.; John Wiley: New York, 2001;Chapter 16.

(24) Johnsson, M.; Bergstrand, N.; Edwards, K.; Stalgren, J.J. R. Adsorption of a PEO-PPO-PEO triblock copolymer onsmall unilamellar vesicles: Equilibrium and kinetic proper-ties and correlation with membrane permeability. Langmuir2001, 17, 3902-3911.

(25) Silvander, M. Steric stabilization of liposomes-A review.Prog. Colloid Polym. Sci. 2002, 120, 25.

(26) Washington, C.; King, S. M.; Heenan. R. H. Structure ofblock copolymers adsorbed to perfluorocarbon emulsions. J.Phy. Chem. 1996, 100, 7603-7609.

(27) Hato, M.; Minamikawa, H.; Tamada, K.; Baba, T.; Tanabe,Y. Self-assembly of synthetic glycolipid/water systems. Adv.Colloid Interface Sci. 1999, 80, 233-270.

(28) Smith, P. F. Archaebacteria and other specialized bacteria.In Microbial Lipids; Ratledge, C., Wilkinson, S. G., Eds.;Academic Press: New York, 1988; Vol. 1, Chapter 8.

(29) Kates, M. Glyco-, phosphoglyco and sulfoglycoglycerolipidsof bacteria. In Handbook of Lipid Research 6, Glycolipids,Phosphoglycolipids and Sulfoglycoglycerolipids; Kates, M. ed.,Plenum Press: New York, 1990; Chapter 1.

(30) Abraham, T.; Hato, M.; Hirai, M. Glycolipid based cubicnanoparticles: Preparation and structural aspects. ColloidsSurf., B 2004, 35, 107-117.

Accepted for publication October 8, 2004.

BP0498544

262 Biotechnol. Prog., 2005, Vol. 21, No. 1