First Direct Observation of Stable Internally Ordered Janus

Letter pubs.acs.org/NanoLett

First Direct Observation of Stable Internally Ordered Janus Nanoparticles Created by Lipid Self-Assembly Nhiem Tran,*,†,‡ Xavier Mulet,† Adrian M. Hawley,‡ Charlotte E. Conn,∥ Jiali Zhai,† Lynne J. Waddington,§ and Calum J. Drummond*,∥ †

CSIRO, Manufacturing Flagship, Clayton, Victoria 3168, Australia SAXS/WAXS Beamline, Australian Synchrotron, Clayton, Victoria 3168, Australia § CSIRO, Manufacturing Flagship, Parkville, Victoria 3052, Australia ∥ School of Applied Sciences, College of Science, Engineering and Health, RMIT University, Melbourne, Victoria 3000 Australia ‡

S Supporting Information *

ABSTRACT: We present the first observation of Janus nanoparticles consisting of stable, coexisting ordered mesophases in discrete particles created by lipid selfassembly. Cryo-TEM images provided visual identification of the multicompartment Janus nanoparticles and, combined with SAXS data, confirmed the presence of mixed cubic phases and mixed cubic/hexagonal phases within individual nanoparticles. We further investigated computer visualization models to interpret the potential interface between the interconnected coexisting nanostructured domains within a single nanoparticle.

KEYWORDS: lipid self-assembly, Janus nanoparticles, cubosome, hexosome, polymorphism, lyotropic liquid crystal he design and fine control of nanoscale structure represents a major technological challenge, necessary for further development of advanced multifunctional materials. Janus nanoparticles, with dual coexisting phases, have been engineered for inorganic and polymeric materials and found application, for example, as nano probes to study interactions between the nanoparticle and the cell.1−3 However, the observation of stable, lipid-based Janus nanoparticles has not yet been recorded.4 Transient multicompartment lipid particles were briefly observed by Angelov et al. within protein−lipid nanoparticles5 at an early stage of particle formation. In this study, we report the coexistence of stable, multiple ordered lyotropic liquid crystalline phases within a discrete Janus-type lipid particle. Self-assembled lipid nanoparticles are finding increased application in a range of biomedical and nanotechnological applications, predominantly in the fields of drug or siRNA delivery, nanoparticle templating and membrane protein crystallization, with further prospective uses as biosensors and biofuel cells.6−19 Their flexible structure can adapt to accommodate bioactive molecules while their large internal surface area (up to 400 m2 g−1)20 and aqueous pore networks provide a diffusion matrix for controlled release21 and biosensing applications. 22 This is particularly true for cubosomes and hexosomes, nanoparticulate dispersions of ordered inverse cubic and hexagonal lyotropic liquid crystalline lipid mesophases. They are thermodynamically stable in excess

T

© XXXX American Chemical Society

water over prolonged periods and can be biocompatible.23 They can also be functionalized via the introduction of additives useful for active targeting in drug delivery and for grafting the cubic phase onto a solid substrate for biosensor applications.19 However, we have, to date, been restricted to a single lipid mesophase with a single set of properties within a nanoparticle, limiting their potential for applications such as drug delivery. The engineering of a Janus bifeature into the well characterized lipid nanoparticle system provides the opportunity to create a wealth of beneficial controllable properties, including the encapsulation of bioactive molecules with varying sustained release profiles. The Janus nanoparticle dispersions developed herein were based on the lipid monoolein (MO) and sterically stabilized using Pluronic F127. This lipid system is well characterized and utilized in membrane protein crystallization and drug delivery. The internal nanostructure of the particles was controlled via the addition of a known amount of capric (decanoic) acid (CA).23,24 MO and CA are generally regarded as safe ensuring the nanoparticles broad applicability. A partial phase diagram for MO-CA-F127-H2O dispersions was determined by using high-throughput synchrotron small angle X-ray scattering Received: May 4, 2015 Revised: May 15, 2015

A

DOI: 10.1021/acs.nanolett.5b01751 Nano Lett. XXXX, XXX, XXX−XXX

Letter

Nano Letters

Figure 1. Phase diagram of MO-CA nanoparticles and identification of SAXS profiles of mixed-phase nanoparticles. Phase diagram at different temperatures (a) with identified phases: QIIP (◇), QIID (+), HII (○), and EME (×). The phase diagram was plot against the CA:MO (wt:wt) ratios in log scale. One dimensional SAXS profiles of QIIP/QIID (b) and QIID/HII (c) mixed-phase nanoparticles with corresponding assigned Braggs peaks. QIIP and QIID peaks are identified using solid and dotted arrows, respectively (b). The peaks are indexed by plotting q(hkl) against ν = (h2 + k2 + l2)1/2 where h, k, and l are Miller indices (Inset b, QIIP (red ◆), QIID (green +)). Open symbols in (Inset b) (red ◇) show reflections allowed by the space group but not visible in the scattering profile. HII and QIID peaks are identified using solid and dotted arrows, respectively (c). The peaks are indexed by plotting q(hkl) against ν = (h2 + k2 + l2)1/2 for cubic phase and ν = (h2 + k2 + hk)1/2 for hexagonal phase (Inset c, QIID (green +), HII (blue ●)).

at higher CA concentration (Figure 1a). Although the Gibbs Phase Rule suggests that a quaternary system of MO-CA-F127H2O can have up to four coexisting phases, no such four-phase region was observed in the phase diagram. The lattice parameters of each identified phase in the phase diagram and one-dimensional scattering profiles are presented in Supporting Information Table S1 and Figure S1, respectively. Compositions selected for further analysis included samples with CA:MO = 0.06 (wt:wt) (QIIP/QIID coexistence) and CA:MO = 0.12 (wt:wt) (QIID /HII coexistence) (Figure 1b,c). A combination of cryogenic transmission electron microscopy

(SAXS) (Figure 1a) to identify multiphase regions of interest. As the CA content increased, various mesophases were observed including the inverse bicontinuous cubic phase of space group Im3m (primitive, QIIP) and Pn3m (diamond, QIID), the inverse hexagonal phase (HII), and an emulsified microemulsion (EME). Consistent with the Gibbs Phase Rule, mixed-phase regions are more prevalent in the phase diagram of ternary and quaternary systems and several two-phase coexistence regions were observed. These included mixed QIIP and QIID cubic mesophases at low concentration of CA, and a QIID cubic phase coexisting with a hexagonal (HII) phase B

DOI: 10.1021/acs.nanolett.5b01751 Nano Lett. XXXX, XXX, XXX−XXX

Letter

Nano Letters

Figure 2. Cryo-TEM images and fast Fourier transformation of Janus nanoparticles. Mixed QIIP/QIID (a) and QIID /HII (b) phases in the same nanoparticle are presented.

cubic symmetry depending on the orientation of the particle.26 In this case, phase differentiation was straightforward as the hexagonal region showed the typical “fingerprint” patterns, whereas the cubic region is associated with an unambiguous cubic symmetry. FFT analysis of the cubic region revealed twodimensional cubic symmetry of the nanoparticle viewed from [112] direction with {110} and {111} reflections visible. The {111} reflection confirms that the cubic phase is QIID as this reflection is forbidden in other cubic phases. The distance between the {110} planes was 6.3 nm and the distance between the {111} planes was 5.3 nm, giving a lattice parameter of 8.9 nm in good agreement with the SAXS data. FFT analysis of the hexagonal region gave a tube-center-to-center distance of 5.6 nm. Single and mixed-phase nanoparticles were both observed via cryo-TEM, indicating that three populations of nanoparticles contribute to the mixed SAXS profiles. Further survey of 71 nanoparticles showed that 37 were QIID, 28 were HII, and 6 were mixed QIID/HII. Additional images of possible multiphase nanoparticles of mixed QIID/HII mesophases can be found in Supporting Information Figure S3. We speculate that the existence of multiphase nanoparticles resulted from the formation of domains with distinct coexisting mesophases in the bulk lipid−water system prior to dispersion by sonication. Fragmentation of the domain structure through sonication could result in three distinct particle populations: two single mesophase and one mixed mesophase. The relative population size of the mixed mesophase nanoparticles would depend on the original domain size, which governs the magnitude of the interfacial area between coexisting mesophases. This rationalizes the low number of multiphase nanoparticles compared with single-phase nanoparticles observed in the current study. Furthermore, it should be noted that in this study, the presence of multiple coexisting phases within a single nanoparticle has been observed exclusively in nanoparticles larger than 200 nm. Dynamic light scattering gave average sizes of the cubic and hexagonal nanoparticles of 176 and 155 nm, respectively (data not shown). However, in cryo-TEM data, nanoparticles displaying two phases are consistently larger than this average. We have not yet determined whether a nanoparticle size threshold exists,

(cryo-TEM) and SAXS analysis was used to determine if the multiple phases coexist within the same nanoparticle. Multiphase nanoparticles of mixed QIIP/QIID mesophases (Figure 2a) and mixed QIID /HII mesophases (Figure 2b) were observed via cryo-TEM. The nanoparticles were left at room temperature for at least 2 weeks prior to cryo-TEM analysis. In both case, two regions of different lattice size with a distinct interface are clearly identified. For the QIIP/QIID nanoparticle, fast Fourier transform (FFT) analysis showed two-dimensional hexagonal symmetry in both regions. This is consistent with a projection of the cubic phase along the ⟨111⟩ direction, confirmed by SAXS data. In this symmetry, only the {110} reflections were visible in FFT graphs. For the QIIP and QIID phases, the distances between the {110} planes d110 for each region are 8.7 and 6.86 nm, respectively (Figure 2a). The FFT calculated lattice parameters are 12.3 nm for QIIP and 9.7 nm for QIID and were in good agreement with the corresponding lattice parameters measured by SAXS. Figure 2a also shows a single phase particle with cubic symmetry. Reflections from {110} and {200} planes are visible with d110 and d200 of 8.9 and 6.17 nm, respectively. The FFT analysis results in lattice parameter of 12.5 nm consistent with that of a QIIP cubic phase. From our observation, the majority of the nanoparticles appeared to contain a single phase of either QIIP or QIID. Using cryo-TEM and FFT analysis, we identified and surveyed 226 nanoparticles in the sample that exhibited mixed cubic phase in SAXS profiles. Among those, 123 were identified as QIIP, 81 were QIID, and more importantly, 22 nanoparticles were found to have mixed phases of QIIP and QIID. The sample was formulated and analyzed on two different occasions and the mixed-phase nanoparticles were consistently observed in cryoTEM images. Additional representative images of multiphase nanoparticles of mixed QIIP/QIID mesophases can be found in Supporting Information Figure S2. A clear mixed-phase nanoparticle containing a hexagonal and a cubic phase is presented in Figure 2b. It can be challenging to identify a QIID/HII mixed phase using cryo-TEM as HII nanoparticles can exhibit either a hexagonal symmetry or a typical “finger-print” pattern,25 whereas the cubic phase nanoparticles can also present either hexagonal symmetry or C

DOI: 10.1021/acs.nanolett.5b01751 Nano Lett. XXXX, XXX, XXX−XXX

Letter

Nano Letters

Figure 3. Models of possible mixed-phase nanoparticles. QIIP/QIID mixed cubic phase nanoparticle is presented in (a) and (b). The water channels (rods) (a) and lipid bilayers (b) are continuous between QIIP and QIID phase. A similar model of a QIID/HII mixed-phase nanoparticle is presented in (c, d). The water channels (rods) (c) and lipid bilayers (d) in HII are connected to those in QIID along the [111] direction.

QIIP and QIID via rhomboheral intermediates,30 we developed computer visualizations of such coexistence within a single nanoparticle (Figure 3). The model was based on the assumption that the two phases are symmetrically connected along the common [111] direction and that the catenoidal minimal surfaces of QIIP and QIID are continuous at the mesophase interfaces.30 The model showed that water channels, represented by rods in Figure 3a, run continuously from QIIP to QIID and the bicontinuous nature of both QIIP and QIID phases were preserved. However, the ratio of lattice parameters in our model, a(P)/a(D), was 0.5 and does not match the Bonnet ratio. If the two phases are connected in this fashion, there should be a resizing step at the interface similar to what Squires et al. predicted.29 Additionally, in our model, both phases are viewed from [110] direction. Further inspection of the mixed-phase nanoparticles under cryo-TEM revealed multiple directions at which the two mesophase connected (Supporting Information Figure S2), suggesting nontrivial and nonsymmetrical mesophase interfaces. A similar model was developed for the QIID/HII mixed-phase nanoparticle. Here, the extension of lipid bilayers and water channels in the QIID phase could form the hexagonal packed channels of the HII phase (Figure 3c,d). To our knowledge, this is the first report on the coexistence of multiple stable mesophases within a discrete self-assembled lipid nanoparticle. At this stage, the developed interface models only partially satisfy the geometrical requirements of coexisting phases. In order to advance our understanding of the nanoarchitecture at the phase interface, extensive cryo-TEM tomography may be needed to provide a three-dimensional reconstruction of the particles. The existence of stable multicompartment nanoparticles is thermodynamically feasible,

below which phase coexistence is not stable. However, we consider that through the existence of the initial domains, a larger nanoparticle would be more likely to capture the domain interface and thus is more readily found to contain multiple mesophases. Stable, mixed-phase nanoparticles raise questions regarding the nanoarchitecture at the interface of the two coexisting phases. It is important to determine whether the two mesophases are continuous both via the connected water channels and the lipid bilayers or whether they are completely separated with disconnected water channels. To visualize this coexistence within a single nanoparticle, assuming a continuous connection between the two phases, we have generated computer visualizations of the two mixed-phase regions within the MO-CA-F127-H2O nanoparticulate phase diagram. Two cubic phases coexisting in excess water under equilibrium conditions are theoretically interrelated by the Bonnet transformation. This isometrically maps the underlying minimal surfaces onto each other so that all angles, distances, and areas at all points on the surface are preserved.27,28 The Bonnet ratio predicts the ratio of lattice parameters for two coexisting cubic phases under excess water conditions: a(P)/a(D) = 1.28. Previously, Squires et al. have developed a pictorial representation of the process based on these earlier models.29 They proposed that the phase transformation from QIIP to QIID occurs via three processes. Initially the 6-fold water channel junction in QIIP is stretched along the [111] direction into two 4-fold junctions of QIID. The resultant QIID phase must be stretched by a factor of 2 and rotated by 60° about the [111] axis. On the basis of the “Squires model”29 and the theory of infinite periodic minimal surfaces for a transformation between D

DOI: 10.1021/acs.nanolett.5b01751 Nano Lett. XXXX, XXX, XXX−XXX

Letter

Nano Letters

(3) Lattuada, M.; Hatton, T. A. Nano Today 2011, 6 (3), 286−308. (4) Walther, A.; Müller, A. H. Chem. Rev. 2013, 113 (7), 5194−5261. (5) Angelov, B.; Angelova, A.; Filippov, S. K.; Drechsler, M.; Štěpánek, P.; Lesieur, S. ACS Nano 2014, 8, 5216−5226. (6) Koltover, I.; Salditt, T.; Rädler, J. O.; Safinya, C. R. Science 1998, 281 (5373), 78−81. (7) Leal, C.; Bouxsein, N. F.; Ewert, K. K.; Safinya, C. R. J. Am. Chem. Soc. 2010, 132 (47), 16841−16847. (8) Mulet, X.; Boyd, B. J.; Drummond, C. J. J. Colloid Interface Sci. 2013, 393, 1−20. (9) Fraser, S. J.; Mulet, X.; Martin, L.; Praporski, S.; Mechler, A.; Hartley, P. G.; Polyzos, A.; Separovic, F. Langmuir 2011, 28 (1), 620− 627. (10) Yaghmur, A.; Glatter, O. Adv. Colloid Interface Sci. 2009, 147, 333−342. (11) Fong, W.-K.; Hanley, T.; Boyd, B. J. J. Controlled Release 2009, 135 (3), 218−226. (12) Boyd, B. J.; Whittaker, D. V.; Khoo, S.-M.; Davey, G. Int. J. Pharma. 2006, 309 (1), 218−226. (13) Boyd, B. J.; Whittaker, D. V.; Khoo, S.-M.; Davey, G. Int. J. Pharma. 2006, 318 (1), 154−162. (14) Negrini, R.; Mezzenga, R. Langmuir 2012, 28 (47), 16455− 16462. (15) Angelova, A.; Ollivon, M.; Campitelli, A.; Bourgaux, C. Langmuir 2003, 19 (17), 6928−6935. (16) Angelova, A.; Angelov, B.; Mutafchieva, R.; Lesieur, S.; Couvreur, P. Acc. Chem. Res. 2010, 44 (2), 147−156. (17) Nazaruk, E.; Smoliński, S.; Swatko-Ossor, M.; Ginalska, G.; Fiedurek, J.; Rogalski, J.; Bilewicz, R. J. Power Sources 2008, 183 (2), 533−538. (18) Conn, C. E.; Darmanin, C.; Sagnella, S. M.; Mulet, X.; Greaves, T. L.; Varghese, J. N.; Drummond, C. J. Soft Matter 2010, 6 (19), 4828−4837. (19) Zhai, J.; Scoble, J. A.; Li, N.; Lovrecz, G.; Waddington, L. J.; Tran, N.; Muir, B. W.; Coia, G.; Kirby, N.; Drummond, C. J. Nanoscale 2015, 7, 2905−2913. (20) Drummond, C. J.; Fong, C. Curr. Opin. Colloid Interface Sci. 1999, 4 (6), 449−456. (21) Shah, J. C.; Sadhale, Y.; Chilukuri, D. M. Adv. Drug Delivery Rev. 2001, 47 (2−3), 229−250. (22) Bilewicz, R.; Rowinski, P.; Rogalska, E. Bioelectrochemistry 2005, 66 (1−2), 3−8. (23) Tran, N.; Mulet, X.; Hawley, A. M.; Hinton, T. M.; Mudie, S. T.; Muir, B. W.; Giakoumatos, E. C.; Waddington, L. J.; Kirby, N. M.; Drummond, C. J. RSC Adv. 2015, 5 (34), 26785−26795. (24) Negrini, R.; Mezzenga, R. Langmuir 2011, 27 (9), 5296−5303. (25) Gustafsson, J.; Ljusberg-Wahren, H.; Almgren, M.; Larsson, K. Langmuir 1997, 13 (26), 6964−6971. (26) Sagalowicz, L.; Michel, M.; Adrian, M.; Frossard, P.; Rouvet, M.; Watzke, H.; Yaghmur, A.; De Campo, L.; Glatter, O.; Leser, M. J. Microsc. 2006, 221 (2), 110−121. (27) Larsson, K. J. Phys. Chem. 1989, 93 (21), 7304−7314. (28) Larsson, K.; Tiberg, F. Curr. Opin. Colloid Interface Sci. 2005, 9 (6), 365−369. (29) Squires, A. M.; Templer, R.; Seddon, J.; Woenkhaus, J.; Winter, R.; Narayanan, T.; Finet, S. Phys. Rev. E 2005, 72 (1), 011502. (30) Schröder, G.; Ramsden, S.; Fogden, A.; Hyde, S. Phys. A (Amsterdam, Neth.) 2004, 339 (1), 137−144.

as the Gibbs phase rule predicts up to four coexisting phases for this quaternary system. From here, a thorough investigation of composition, formulation and processing conditions will now be required in order to gain control of the number and size distribution of Janus nanoparticles created by lipid selfassembly. The quest for smarter nanomaterials has driven recent research toward the engineering of nanosystems with controllable functionality. Traditional, isotropic nanoparticles have allowed us to explore the relationship between form and function and are used extensively in many applications. In the future, the need for more complex, biomimetic, multicompartment nanoparticle systems such as Janus nanoparticles will increase. Their application in a wide range of fields including drug delivery, biosensors, nanoelectronics, and catalysis will require further studies on these multicompartment lipid nanoparticles, and these should provide additional insights into this interesting phenomenon.

■

ASSOCIATED CONTENT

S Supporting Information *

Details regarding materials, sample preparation and characterization methods using SAXS and cryo-TEM, analysis of phase and lattice parameters. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.nanolett.5b01751.

■

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Address: CSIRO. Bag 10, Clayton South MDC, VIC, 3169, Australia. *E-mail: [email protected]. Address: RMIT University, 124 La Trobe Street, Melbourne VIC 3000, Australia. Author Contributions

N.T., X.M., and C.J.D. designed experiments. N.T. prepared the samples and analyzed the data. N.T. and A.M.H. performed synchrotron SAXS screening experiments. X.M., C.E.C., and C.J.D. provided intellectual discussions. L.J.W. performed cryoTEM experiments. N.T. and J.Z. generated theoretical models of Janus nanoparticles. N.T., X.M., C.E.C., and C.J.D. wrote the paper. All authors commented on the manuscript. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The authors would like to thank Professor Stephen Hyde, Professor Gerd Schröder-Tusk, and Dr. Benjamin Muir for valuable discussions and support regarding the theoretical model of the mixed-phase nanoparticles. The authors are grateful for the assistance provided by Dr. Stephen Mudie and Dr. Nigel Kirby at the SAXS/WAXS beamline, Australian Synchrotron. N.T. is supported by a SIEF John Stocker Postdoctoral Fellowship. J.Z. is supported by a CSIRO OCE Postdoctoral Fellowship. This research includes work undertaken on the SAXS/WAXS beamline at the Australian Synchrotron, Victoria, Australia.

■

REFERENCES

(1) Du, J.; O’Reilly, R. K. Chem. Soc. Rev. 2011, 40 (5), 2402−2416. (2) Jiang, S.; Chen, Q.; Tripathy, M.; Luijten, E.; Schweizer, K. S.; Granick, S. Adv. Mater. 2010, 22 (10), 1060−1071. E

DOI: 10.1021/acs.nanolett.5b01751 Nano Lett. XXXX, XXX, XXX−XXX