Report

Self-Assembly of Janus Dendrimers into Uniform Dendrimersomes and Other Complex Architectures

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Science  21 May 2010:
Vol. 328, Issue 5981, pp. 1009-1014
DOI: 10.1126/science.1185547

Abstract

Self-assembled nanostructures obtained from natural and synthetic amphiphiles serve as mimics of biological membranes and enable the delivery of drugs, proteins, genes, and imaging agents. Yet the precise molecular arrangements demanded by these functions are difficult to achieve. Libraries of amphiphilic Janus dendrimers, prepared by facile coupling of tailored hydrophilic and hydrophobic branched segments, have been screened by cryogenic transmission electron microscopy, revealing a rich palette of morphologies in water, including vesicles, denoted dendrimersomes, cubosomes, disks, tubular vesicles, and helical ribbons. Dendrimersomes marry the stability and mechanical strength obtainable from polymersomes with the biological function of stabilized phospholipid liposomes, plus superior uniformity of size, ease of formation, and chemical functionalization. This modular synthesis strategy provides access to systematic tuning of molecular structure and of self-assembled architecture.

Biological membranes are complex systems assembled from phospholipids and stabilized by cholesterol, proteins, and carbohydrates (1). They are equipped with nanoscale machinery that includes protein channels to mediate and control electron and proton transfer between the cell and its environment. Liposomes (2), vesicles assembled from natural or synthetic amphiphiles, can mimic biological membranes (1, 36), probe cell machinery (3, 6), and can be configured into biomimetic materials for nanomedicine and other applications (79). Designing vesicles endowed with specific functional features (including tunable mechanical strength, targeted size, uniform size distributions, and tailored transport properties) presents a formidable challenge. Both synthetic (1016) and natural (16, 17) vesicles are typically polydisperse and unstable over time, necessitating tedious fractionation (1417) and stabilization (8, 9, 1821), because specific applications are decided by these factors. Here we report the preparation of a broad class of synthetic amphiphiles known as Janus dendrimers (22) and demonstrate facile self-assembly in water of stable bilayer vesicles referred to as dendrimersomes. Most of these dispersions contain populations of bilayer capsules remarkably uniform in size. We document superior mechanical properties and impermeability to encapsulated compounds. Dendrimersomes can also incorporate pore-forming proteins, coassemble with structure-directing phospholipids and block copolymers, and offer a molecular periphery suitable for further chemical functionalization. This report provides the first description of the preparation, structure, and properties of dendrimersomes (and other select complex dispersions) discovered by screening extensive libraries of Janus dendrimers prepared with exacting chemical fidelity.

Dendrimers are technologically advanced synthetic compounds with monodisperse and precisely branched molecular architectures (2224). Many factors control the ultimate properties and applications of these fascinating materials, including the number of primary branches that emanate from a common linking point, the density of radial branches, and the distribution of chemical moieties located inside the molecular labyrinth or at the periphery of the molecule. A Janus dendrimer is formed by linking two chemically distinct dendritic building blocks, thereby breaking the roughly spherical symmetry that characterizes most dendrimers (2224). When synthesized with judiciously tailored hydrophilic and hydrophobic elements, Janus dendrimers can function as powerful structure-directing amphiphiles, with greater versatility than simple lipids, surfactants, or block copolymers.

Screening libraries of compounds with systematically varying molecular structure and supramolecular architecture represents one of the most powerful contemporary methods for discovering new materials (25). In our experiment, 11 distinct libraries containing a total of 107 uncharged or positively charged amphiphilic Janus dendrimers were synthesized and screened for supramolecular assembly in water. These Janus dendrimers (Fig. 1) were designed from AB3 and constitutionally isomeric AB2 building blocks, incorporating both hydrophilic and hydrophobic segments that can be rapidly combined to produce diverse sets of exact and monodisperse structures (26). Janus dendrimers were prepared with a combination of convergent approaches for the hydrophobic parts, and divergent or convergent methods for the hydrophilic parts (schemes S1 to S16) (26). Two hydrophobic segments (one aliphatic and one mixed aliphatic-aromatic) and six hydrophilic segments derived from oligoethylene oxide, dimethylolpropionic acid, glycerol, thioglycerol, tert-butylcarbamate, and quaternary ammonium salts were combined to yield the portfolio of compounds illustrated in Fig. 1. This simple modular concept allows the fractions of the hydrophilic and hydrophobic portions of the molecule to be varied systematically. Such synthetic versatility and control over molecular architecture and functionality make Janus dendrimers competitive with traditional surfactants and diblock copolymers (917).

Fig. 1

Libraries of amphiphilic Janus dendrimers.

All of the Janus dendrimers shown in Fig. 1 can be dispersed by injection (17) from dilute solution (typically 10 to 20 mg/mL in ethanol, but also tetrahydrofuran, acetone, or dimethyl sulfoxide) into water. The resulting particle dispersions were characterized for basic morphology by cryogenic transmission electron microscopy (cryo-TEM) and sorted into two categories: (i) dendrimersome (vesicle)–forming assemblies and (ii) more complex supramolecular assemblies. The wide spectrum of supramolecular architectures that we observed illustrates the unique self-assembly characteristics of Janus dendrimers.

Figure 2, A and B, illustrates a cryo-TEM image of small spherical dendrimersomes derived from (3,4)12G1-PE-(3,5)-3EO-G1-(OCH3)4 (library 2 in Fig. 1). Similar images were obtained from numerous other Janus dendrimers, each exhibiting a unilamellar domain boundary with bilayer thicknesses ranging from 5 to 8 nm (table S4). A notable feature of this picture is the narrow range of dendrimersome sizes. To more quantitatively establish the size, polydispersity, and stability of the dendrimersomes, we characterized aqueous dispersions by dynamic light scattering as a function of concentration, temperature, and time. A typical ethanol solution (10 mg/mL) injection into water (2 mL, final concentration = 0.5 mg/mL) produced dendrimersomes with mean radii of 33 to 732 nm (figs. S15 to S21) and polydispersities (normalized second cumulants) mostly ranging from 0.02 to 0.20 (figs. S15 to S21) with an upper limit of 0.53 (here, 0 corresponds to a perfectly uniform size distribution). Some of these assemblies were stable for long periods of time at room temperature or even when annealed from 22° to 80°C (figs. S2 and S3). Dendrimersomes derived from library 1 showed a dependence of both size and polydispersity on the concentration in ethanol before injection (table S2), whereas those associated with libraries containing oligoethyleneoxide in the hydrophilic part of the molecule (for example, library 2) were quite insensitive to these parameters.

Fig. 2

(A) Cryo-TEM of dendrimersomes from (3,4)12G1-PE-(3,5)-3EO-G1-(OCH3)4 in ultrapure water formed by ethanol injection and (B) their 3D intensity profile. (C) Fluorescence microscopy image of a giant dendrimersome assembled from (3,5)12G1-PE-(3,4)3EO-(OH)4, encapsulating both hydrophobic Nile red and hydrophilic calcein dyes. (D) Microscopy image and (F) 3D intensity profile of giant dendrimersome from (3,4)12G1-PE-BMPA-G2-(OH)8 visualized with Nile red. (E) Giant dendrimersome from (3,4,5)12G1-PE-(3,4,5)3EO-G1-(OH)6 visualized with Nile red and calcein. (G) Micropipette aspiration assessment of mechanical strength by micro deformation under negative pressure of a (3,5)12G1-PE-BMPA-G2(OH)8 giant dendrimersome. (H) The same dendrimersome under negative pressure showing small deformation of the membrane. White arrow indicates the deformation of the membrane under micropipette suction pressure. (I) Areal strain determined from micropipette aspiration upon rupture of the same dendrimersome (αc). The asterisk indicates the point of membrane failure at the critical areal strain.

Giant dendrimersomes, 2 to 50 μm in diameter, were prepared by hydration experiments with ultrapure water or phosphate-buffered saline. Giant dendrimersomes provide access to measurements complimentary (27) to those performed by cryo-TEM on small dendrimersomes. These experiments are required to establish the mechanical integrity and permeability of the dendrimersomes, as well as for comparison of these critical features with liposomes (26) and polymersomes (1013, 26). In a typical formulation, a 200-μL aliquot of Janus-dendrimer solution in methylene chloride (10 mg/mL) was uniformly deposited on the surface of a roughened Teflon (DuPont, Wilmington, DE) plate, placed in a vial, and then the solvent evaporated over the course of 12 hours. Addition of water and hydration at 60°C for 12 hours results in the formation of giant unilamellar dendrimersomes. Addition of a molecular dye (for instance, calcein) or drug (e.g., doxorubicin) (7) during the hydration step permits encapsulation within the dendrimersomes during self-assembly.

Visualization of both the wall and cavity of giant dendrimersomes was accomplished by fluorescence microscopy after encapsulation of hydrophobic (Nile red) and hydrophilic (calcein) dyes. We observed the hydrophobic dye to localize exclusively in the wall, whereas the hydrophilic dye was noted only in the aqueous interior (Fig. 2, C to F). Micromanipulation experiments with the use of an established micropipette aspiration technique (Fig. 2, G to I) (26) revealed robust mechanical stability: The areal expansion moduli can be tuned over a wide range, 42 ≤ Ka ≤ 976 mN/m (table S5), with lipidlike critical areal strains, 0.03 ≤ αc ≤ 0.06 (Ka, areal expansion modulus; αc, critical areal strain). The strongest dendrimersomes outperform unmodified liposomes by a wide margin (Ka ≤ 234 mN/m) and are competitive with cholesterol-stabilized liposomes [Ka = 781 and 1286 mN/m with 50 and 78% loading of cholesterol in 1-stearoyl-2-oleoyl phosphatidylcholine (SOPC), respectively] (26). Dendrimersomes are considerably stronger, although less compliant, than the toughest polymersomes (Ka ≤ 140 mN/m and αc ≥ 0.19) (10).

The thickness, impermeability, and mechanical properties of giant dendrimersomes indicate that they are excellent candidates for models of biological membranes (1, 3, 6, 13, 16, 18, 19, 27). A membrane thickness of 5 to 8 nm (table S4) should be ideal for incorporating pore-forming proteins, such as melittin (28). This has proven difficult to achieve with polymersomes (13) due to greater membrane thicknesses (generally >8 nm). Melittin incorporation into dendrimersomes was investigated with Janus dendrimers from library 1 and a phospholipid (SOPC) control liposome formed by standard film hydration (figs. S11 and S12). Dendrimersomes containing the fluorescent dye 1-aminonaphthalene-3,6,8-trisulfonate (ANTS) and the quencher α,α′-dipyridinium p-xylene dibromide, which quenches ANTS fluorescence at high concentrations (26), were prepared by film-hydration experiments. After the addition of melittin, a dramatic increase in ANTS fluorescence was observed, which is associated with the release of the dye due to melittin-induced pore formation. Subsequent lysis of the vesicles with Triton-X revealed that 60% of the dye was released after melittin incorporation (fig. S11). This experiment demonstrates that, although dendrimersomes have the stability and mechanical properties found with polymersomes, they simultaneously exhibit the structure and function of stabilized phospholipid liposomes.

Dendrimersome stability with time was investigated in biologically relevant media by the formation of membranes via ethanol injection into both citrate buffer and phosphate-buffered saline. Those from library 1 are stable in citrate buffer over a period of 2 weeks and in ultrapure water up to at least 244 days at room temperature (fig. S3). Dendrimersomes from library 2 exhibit excellent stability in both ultrapure water and citrate and phosphate buffers. Selected dendrimersomes from libraries 1 and 2 were loaded with the anticancer drug doxorubicin (7, 8) by hydration of Janus-dendrimer films. Negligible release was observed at physiological temperature and pH (~7.2 to 7.4), again demonstrating the impermeability of dendrimersomes (fig. S13). Predictably, under acidic conditions (pH ~ 5.2 to 5.4), the aromatic-aliphatic ester groups of the dendrimersome cleave, resulting in release of the drug (fig. S13), thereby suggesting pathways to engineer these encapsulants for targeted intracellular drug delivery (8, 9).

Collectively, these results provide powerful evidence that Janus dendrimers produce bilayer vesicles with morphologies and properties competitive with the most advanced liposomes and polymersomes. Architecturally, this category of amphiphiles is qualitatively different than classical lipids or block copolymers. Self-assembly of amphiphilic compounds is a complex subject that remains the target of extensive experimentation and theory. Conceptually, diblock copolymers are most easily understood because the formation of bilayer vesicles, cylindrical micelles, and spherical micelles is rooted in statistically averaged properties (29). A specific morphology reflects the thermodynamically most stable interfacial curvature, which is governed by a competition between interfacial tension and the volume occupied by the hydrophobic and hydrophilic blocks. Interfacial packing can be crudely modeled with the use of simple geometric objects, such as wedges or cones, that account for molecular space filling. This geometric approach also is widely applied to lipids and surfactants (30), which form predominately bilayer vesicles, spherical micelles, and occasionally cylindrical micelles. Tinkering with the shape and size of a lipid or block copolymer influences the spontaneous curvature and, hence, the morphology. Unlike the uniformly sized dendrimersomes reported here, lipids and surfactants rarely produce low polydispersity vesicles, and diblock copolymers never do by simple injection (14, 15). Jung et al. (31) have reported that a large bending modulus and a spontaneous curvature are both required to obtain a population of uniform vesicles at equilibrium. This has been achieved only with certain surfactant mixtures.

Controlled branching introduces a powerful new factor in the design of self-assembling amphiphiles. Hydrophobic and hydrophilic chemical functionality can be precisely positioned within Janus dendrimers, resulting in unprecedented control over molecular shape and surface properties. These compounds contain a rich three-dimensional (3D) molecular structure that is manifested in the shape, size, strength, and chemical functionality of the resulting assemblies. Selected Janus dendrimers tagged with Texas red dye on the periphery were shown to coassemble into fluorescent giant unilamellar dendrimersomes with unlabeled Janus dendrimers, block copolymers, and phospholipids (scheme S17 and figs. S7 to S10). This indicates the potential utility of tagged Janus dendrimers in theranostics for detection and treatment of diseases.

Application of conventional geometric models to the Janus dendrimers is unwarranted given the molecular complexity of these compounds. To gain insight into the self-assembly process, we have therefore employed computer simulations based on coarse-grained (CG) molecular dynamics (32). For example, this approach was applied to Janus dendrimers (3,5)12G1-PE-BMPA-G1-(OH)4 and (3,5)12G1-PE-BMPA-G2-(OH)8 known to form dendrimersomes on the basis of cryo-TEM. CG interaction potentials were derived from fits to all-atom simulations of the Janus-dendrimer bilayers. Figure 3 displays snapshots of the resulting self-assembly process, beginning with the disordered state and culminating with the formation of a dendrimersome on a nanosecond time scale. These computer simulations therefore reinforce the interpretation of the experiments.

Fig. 3

(A) Molecular model used for the self-assembly Janus dendrimer (3,5)12G1-PE-BMPA-(OH)4. (B) Self-assembly of (3,5)12G1-PE-BMPA-(OH)4 using CG molecular dynamics. The CG interaction potentials were derived from fitting to an all-atoms simulation of dendrimer bilayer. Spontaneous bilayer structure formation occurs on a multi-nanosecond time scale using a relatively small number of amphiphiles in the simulation box. The initial snapshot, showing isotropic mixing of dendrons at t = 0 ns, is shown here. (C) Snapshot of the simulation at t = 20 ns showing lamellar structure formation. (D) Complete simulation showing spontaneous formation of bilayer at t = 40 ns. (E) Complete simulation showing spontaneous vesicle formation at t = 80 ns. (F) Cut-away view of (E), showing the hollow core of the vesicle. The solvent has been removed for visual clarity. Red, hydroxyl; black, hydrophobic fragment of 2,2-bis(hydroxymethyl) propionate; dark purple, ester; magenta, neopentyl fragment; gray, benzene ring fragment; brown, dioxybenzene ring fragment; green, alkane fragment; blue, terminal alkyl.

Additional evidence supporting the contention that Janus dendrimers are qualitatively different than conventional surfactants and diblock copolymers may be found in other structures illustrated in cryo-TEM images derived from several compounds listed in Fig. 1. Figure 4 illustrates a collection of nanoscale morphologies selected from many that we have documented. Figure 4 (along with Fig. 2A) illustrates sensitivity to systematic structural variations within library 2. Close inspection of Fig. 4A [(3,4)12G1-PE-(3,4)-3EO-G1-(OMe)4] reveals faceted, polygonal (33) dendrimersomes, which probably reflects an intriguing compromise between amphiphile-driven bilayer formation and molecular rigidity associated with the branched core. Figure 4B [(3,4)12G1-PE-(3,4)-2EO-G1-(Me)4] shows particles known as cubosomes (34), which contain a bicontinuous internal morphology. Disklike (35) and toroidal dispersions are found within Fig. 4I [(3,4,5)12G1-PE-(3,5)-3EO-(OMe)4], whereas (3,5)12G1-PE-(3,4,5)-3EO-(OMe)6 generates tubular dendrimersomes (Fig. 4G) (36). Two final examples, drawn from library 2 [(3,4,5)12G1-PE-(3, 5)-3EO-(OMe)6] and library 8 [tris12-PE-BMPA-G2-(OH)8] are presented in Fig. 4, C and H, evidencing spherical micelles and flat, helical (twisted) ribbons, respectively. Course-grained molecular dynamics computer simulation is capable of capturing elements of this complexity, as illustrated in Fig. 5. These images demonstrate spontaneous self-assembly of (3,5)12G1-PE-BMPA-(OH)4 into bicontinuous and disklike objects, depending on the level of hydration, with marked resemblance to morphologies found in Fig. 4, B, E, I, and L.

Fig. 4

Cryo-TEM and 3D intensity profiles of (A and D) polygonal dendrimersomes from (3,4)12G1-PE-(3,4)-3EO-G1-(OMe)4. (B and E) Bicontinuous cubic particles co-existing with low concentration of spherical dendrimersomes from (3,5)12G1-PE-(3,4,5)-2EO-(OMe)6. (C and F) Micelles from (3,4,5)12G1-PE-BMPA-G2-(OH)8. (G and J) Tubular dendrimersomes from (3,5)12G1-PE-(3,4,5)-3EO-(OMe)6. (H and K) Rodlike, ribbon and helical micelles from tris12-PE-BMPA-G2-(OH)8. (I and L) Disklike micelles and toroids from (3,4,5)12G1-PE-(3,5)-3EO-(OMe)4.

Fig. 5

Self-assembly of the Janus dendrimer (3,5)12G1-PE-BMPA-(OH)4 using CG molecular dynamics and a large number of dendrimers. (A) Spontaneous and persistent bicontinuous phase occurs within 200 ns when modeled with a ratio of ~133 water molecules per Janus dendrimer. (B and C) Top view and side view, respectively, of a disklike micelle that assembles within 400 ns when the same dendrimer is modeled in a more dilute system having ~808 water molecules per dendrimer. The solvent has been removed for visual clarity.

Though it would be premature to draw detailed conclusions regarding the relationship between specific molecular variations and resulting self-assembled structure at this time, it is clear that Janus dendrimers provide a rich palette for the engineering of new and exciting materials by self-assembly.

Supporting Online Material

www.sciencemag.org/cgi/content/full/328/5981/1009/DC1

Materials and Methods

SOM Text

Figs. S1 to S21

Schemes S1 to S17

Tables S1 to S8

References

  • Present address: DuPont Central Research and Development, Wilmington, DE 19880, USA.

References and Notes

  1. Materials and methods are available as supporting material on Science Online.
  2. We thank the NSF (grant DMR-0548559), NSF-funded Materials Research Science and Engineering Centers at the University of Pennsylvania and at the University of Minnesota, the Academy of Finland, and the P. Roy Vagelos Chair at the University of Pennsylvania for financial support. We also thank T. Tuttila, N. Kuuloja, and M. Lahtinen from the University of Jyväskylä for contributions on the synthesis and analysis of some Janus dendrimers from library 1. Additional funding was provided by the Finnish Cultural Foundation.
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