Hydrophilic Macroionic Solutions: What Happens When Soluble Ions

Nov 4, 2009 - Log−log plot of the SAXS data for 0.25 mM {Mo72V30} in acetone/water mixed solvents containing various amount of acetone (an effect wh...
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Hydrophilic Macroionic Solutions: What Happens When Soluble Ions Reach the Size of Nanometer Scale? Tianbo Liu* Department of Chemistry, Lehigh University, 6 East Packer Avenue, Bethlehem, Pennsylvania 18015 Received August 6, 2009. Revised Manuscript Received September 23, 2009 Large, hydrophilic inorganic ions (mostly polyoxometalate macroions and cationic metal-organic hybrid nanocages) with high solubility in water and/or other polar solvents demonstrate unique solution behaviors. In dilute solutions, they behave significantly different from small simple ions (as described by the Debye-H€uckel theory) because the macroions cannot be treated as point charges or large, insoluble colloidal suspensions (usually described by the DLVO theory) because the macroions form homogeneous, stable “real solutions”. The size disparity between the macroions and their counterions results in complex macroion-counterion interaction and leads to the self-assembly of macroions into single-layered, hollow, spherical “blackberry” structures. The blackberries, with robust and very stable structures mimicking biological membranes, can adjust their size accurately and reversibly in response to the change of solvent content, charge density on the macroions, or in some cases merely solution pH. The blackberry membrane is permeable to small cations. The inorganic macroions with well-defined size, shape, mass, charge density (even accurately tunable within certain range), and no intramolecular interaction can be treated as simple model systems to understand the intermolecular interaction in polyelectrolyte solutions. The blackberry structures show certain similarities to the spherical virus capsids, from the overall structure to the kinetic properties of formation.

Introduction Understanding complex solution behavior has always been challenging. It becomes even more difficult if the solutes are charged, for example, electrolyte solutions. The most well-known approach for describing simple ionic solutions (such as dilute NaCl aqueous solution) is the Debye-H€uckel theory1 which works well for very dilute ionic solutions, and its extended form can be applied to higher ionic concentrations. On the other hand, large, charged colloidal particles are fundamentally different. They are temporarily suspended in (usually) liquid media with the help of their charges (electrostatic interactions) and are thermodynamically unstable. The widely applied Derjaguin-Landau-Verwey-Overbeek theory (or DLVO theory) attributes the stability of colloids to the competition between the attractive van der Waals forces and repulsive electrostatic interactions.2 Sogami and Ise indicate that in colloidal suspensions there exist long-range weak attractions introduced by counterions.3,4 A key question is how the solutes between simple ions and colloids, in term of size and charge density, behave in solution. Or the question can be presented as “What happens when soluble ions reach the size of nanometer scale?” We can speculate that such large ions (macroions) cannot be described by the Debye-H€uckel theory anymore even in dilute solutions because the macroions cannot be treated as point charges, while on the other hand they are still soluble and form homogeneous “real solutions” which distinguishes them from colloidal particles. *E-mail: [email protected]. (1) Debye, P. J.; H€uckel, E. Phys. Z. 1923, 24, 185. (2) (a) Derjaguin, B. V.; Landau, L. Acta Physicochim. URSS 1941, 89, 555. (b) Verwey, E. J.; Overbeek, J. T. G. Theory of the Stability of Lyophobic Colloids; Elsevier: Amsterdam, 1948. (3) Sogami, I.; Ise, N. J. Chem. Phys. 1984, 81, 6320. (4) Ise, N.; Sogami, I. Structure Formation in Solution; Springer-Verlag: Berlin, Heidelberg, 2005.

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Such macroions also have connections to polyelectrolyte (including biomacromolecular and dendrimeric) solutions. The polyelectrolyte solutions are still very poorly understood,5 mostly due to the coexistence of intermolecular and intramolecular interaction with the latter including variable chain conformations and entanglements, the effect of additional electrolytes, as well as sample polydispersity. The solution systems with well-defined molecular structure (no intramolecular interaction), mass, shape (no polydispersity), charge density (even tunable in a certain range), and no additional salts would be ideal model systems for understanding polyelectrolyte solutions.

Polyoxometalate (POM) Molecular Clusters and Hydrophilic Macroanions The giant polyoxometalate (POM) molecular clusters can nicely meet the above requirements. Recent progress in synthetic inorganic chemistry has produced a large family of such giant clusters, representing some of the largest inorganic molecules known so far.6-10 The POMs are formed by covalently connecting multiple transition metal (Mo, W, V, Cr, Fe, etc.) oxide polyhedrals (used as building blocks) and arranging them into geometrically beautiful molecular clusters (some are nanometers in size). They are structurally well-defined and show a wide variety (5) (a) Sedlak, M.; Amis, E. J. J. Chem. Phys. 1992, 96, 826. (b) Sedlak, M. J. Chem. Phys. 2002, 116, 5256. (c) Rulkens, R.; Wegner, G.; Thurn-Albrecht, T. Langmuir 1999, 15, 4022. (d) Henle, M. L.; Pincus, P. A. Phys. Rev. E 2005, 71, 060801. (e) Mukherjee, A. K.; Schmitz, K. S.; Bhuiyan, L. B. Langmuir 2003, 19, 9600. (f) Schmitz, K. S.; Lu, M.; Singh, N.; Ramsay, D. J. Biopolymers 1984, 23, 1637. (6) Polyoxometalates special issue: Hill, C. L., Ed. Chem. Rev. 1998, 98, 1-387. (7) (a) M€uller, A.; Roy, S. Coord. Chem. Rev. 2003, 245, 153. (b) M€uller, A.; K€ogerler, P.; Dress, A. W. M. Coord. Chem. Rev. 2001, 222, 139. (8) Proust, A.; Thouvenot, R.; Gouzerh, P. Chem Commun. 2008, 1837. (9) Long, D.-L.; Burkholder, E.; Cronin, L. Chem. Soc. Rev. 2007, 36, 105. (10) (a) Parker, W. O.; Millini, R.; Kiricsi, I. Inorg. Chem. 1997, 36, 571. (b) Lee, A. P.; Philips, B. L.; Olmstead, M. M.; Casey, W. H. Inorg. Chem. 2001, 40, 4485. (c) Allouche, L.; Gerardin, C.; Thierry, L.; Ferey, G.; Taulelle, A. F. Angew. Chem., Int. Ed. 2000, 39, 511. (d) Mialane, P.; Dolbecq, A.; Lisnard, L.; Mallard, A.; Marrot, J.; Secheresse, F. Angew. Chem., Int. Ed. 2002, 41, 2398.

Published on Web 11/04/2009

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Invited Feature Article Table 1. Chemical Formulas of Some Large, Hydrophilic POM Molecular Clusters Described in This Article

abbreviation

formula

ref

{Mo154} {Mo132} {Mo72Fe30} {Mo72V30} {Mo72Cr30} {W48P8Cu20} {Mo368} {P4Y8W43}

Na15[Mo154O462H14(H2O)70]0.5[Mo152O457H14(H2O)68]0.5 3 ca.400H2O (NH4)42[{(MoVI)MoVI5O21(H2O)6}12{Mo2O4(CH3COOH)}30] 3 ca.300H2O 3 ca.10CH3COONH4 [MoVI72FeIII30O252(CH3COO)12{Mo2O7(H2O)}2{H2Mo2O8(H2O)}(H2O)91] 3 ca.150H2O Na8K14(VO)2[{(MoVI)MoVI5O21(H2O)3}10{(MoVI)MoVI5O21(H2O)3(SO4)}2 {VIVO(H2O)}20{VIVO}10({KSO4}5)2] 3 ca.150H2O [{Na(H2O)12}⊂{MoVI72CrIII30O252(CH3COO)19(H2O)94}] 3 ca.120H2O K12Li13[Cu20Cl(OH)24(H2O)12(P8W48O184)] 3 22H2O Na48[HxMo368O1032(H2O)240(SO4)48] 3 ca.1000H2O K15Na6(H3O)9[(PY2W10O38)4(W3O14)] 3 9H2O

11 12 13 14 15 16 17 18

Figure 1. Some typical large polyoxometalate molecular clusters (and their sizes) described in the Article. Detailed formulas are listed in Table 1.

of structural, magnetic, and electronic properties. POMs offer unique opportunities in both fundamental studies and practical applications in various fields, for example, as catalysts and antitumor agents. More details can be found in a series of comprehensive reviews published in Chemical Reviews in 1998 (Hill)6 and in some more recent review articles.7-9 Most of large POM clusters are polyoxomolybdates, polyoxotungstates, and their heteropolyoxometalate derivatives. Quite a few types of these large POMs are hydrophilic in nature and are very soluble in polar solvents, mainly due to their surface water ligands coordinated to the metal centers and their inherent charges. Details for the POMs discussed in this Article are provided in Table 1; and their molecular structures are shown schematically in Figure 1. These POMs can be categorized into two groups depending on the nature of their charges: strong electrolytes and weak electrolytes. The POM clusters in the first group carry inherent charges in crystals which are balanced by multiple small cations. In solution, the majority part of the clusters usually exists as macroanions. Some POM macrocations have also been synthesized recently.10 In the second group, the POMs are almost neutral clusters (molecules) in crystals but in aqueous solution their water ligands attached to non-Mo (or nonW) metals can partially deprotonate. As the result, the clusters become negatively charged by carrying certain localized charges. Overall, the clusters behave as weak acids in solution, and their charge density can be controlled by solution pH, which will be described in detail in the following text. (11) (a) M€uller, A.; Das, S. K.; Fedin, V. P.; Krickemeyer, E.; Beugholt, C.; B€ogge, H.; Schmidtmann, M.; Hauptfleisch, B. Z. Anorg. Allg. Chem. 1999, 625, 1187. (b) M€uller, A. M.; Serain, C. Acc. Chem. Res. 2000, 33, 2. (12) M€uller, A.; Krickemeyer, E.; B€ogge, H.; Schmidtmann, M.; Peters, F. Angew. Chem., Int. Ed. 1998, 37, 3360. (13) M€uller, A.; Sarkar, S.; Shah, S. Q. N.; Trautwein, A. X.; Sch€unemann, V. Angew. Chem., Int. Ed. 1999, 38, 3238. (14) Botar, B.; K€ogerler, P.; Hill, C. L. Chem Commun. 2005, 25, 3138. (15) Todea, A. M.; Merca, A.; B€ogge, H.; van Slageren, J.; Dressel, M.; Engelhardt, L.; Luban, M.; Glaser, T.; Henry, M.; M€uller, A. Angew. Chem., Int. Ed. 2007, 46, 6106. (16) (a) Mal, S. S.; Kortz, U. Angew. Chem., Int. Ed. 2005, 44, 3777. (b) Jabbour, D.; Keita, B.; Nadjo, L.; Kortz, U.; Mal, S. S. Electrochem. Commun. 2005, 7, 841. (c) Alam, M. S.; Dremov, V.; M€uller, P.; Postnikov, A. V.; Mal, S. S.; Hussain, F.; Kortz, U. Inorg. Chem. 2006, 45, 2866. (17) M€uller, A.; Beckmann, E.; B€ogge, H.; Schmidtmann, M.; Dress, A. Angew. Chem., Int. Ed. 2002, 41, 1162. (18) Howell, R. C.; Perez, F. G.; Jain, S.; Dew, W. H.; Rheingold, A. L.; Francesconi, L. C. Angew. Chem., Int. Ed. 2001, 113, 4155.

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Characterization of the Self-Assembly of POM Macroanions in Dilute Solutions Most of the above-mentioned POMs are quite soluble in aqueous solution to form clear, stable solutions. For example, the solubility for {Mo154} and {Mo132} clusters is well above 100 mg/mL at room temperature. However, many of these macroions do not exist as discrete ions even in very dilute solutions.19,20 As shown in Figure 2, transmission electron microscopy (TEM) studies indicate that in very dilute {Mo154} aqueous solution there exist many spherical assemblies with relatively uniform size (around 90 nm).19 This is unexpected, as the highly soluble and fully hydrophilic macroanions should not be attracted to each other. More interestingly, during the atomic force microscopy (AFM) study when shifting from conventional mode to vacuum mode the assemblies suddenly burst, suggesting that they might not be solid aggregates.20 A thorough exploration of this phenomenon was achieved by laser light scattering (LLS) studies which include both static and dynamic light scattering (SLS and DLS) measurements. SLS study measures the scattered intensity from solution at different scattering angles and can generate the information on the weightaverage molecular weight (Mw), radius of gyration (Rg), and second virial coefficient (A2) of the solutes. DLS study measures the intensity-intensity time correlation function (then converted to electric field time correlation function) by means of a multichannel digital correlator. The time correlation function, analyzed by the CONTIN method, can provide the information regarding the diffusion coefficients of the solutes. Consequently, information such as the average hydrodynamic radius (Rh) and particle size distribution can be obtained. A combination of SLS and DLS can be very useful in characterizing complex solutions.19 As shown in Figure 3, DLS study on the 0.010 mg/mL {Mo154} aqueous solution at pH=3.0 shows that there are large assemblies with an average Rh of 45 nm and a narrow size distribution. For spherical objects (known from Figure 2a), their average Rh value is equivalent to the overall radius of the sphere. The result is consistent with the TEM study (90 nm size). SLS study analyzed by Zimm plot indicates that the assemblies have an average Mw of ∼(2.54 ( 0.25)  107 g/mol (19) Liu, T.; Diemann, E.; Li, H.; Dress, A.; M€uller, A. Nature 2003, 426, 59. (20) M€uller, A.; Diemann, E.; Kuhlmann, C.; Eimer, W.; Serain, C.; Tak, T.; Kn€ochel, A.; Pranzas, P. K. Chem. Commun. 2001, 1928.

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Figure 2. (left) TEM image on dilute aqueous solution of {Mo154} macroions showing the existence of spherical, ∼45 nm radius assemblies. (right) Zimm plot based on the SLS study of the {Mo154} aqueous solutions at pH = 3; (inset) CONTIN analysis on the DLS study of the same solution. Reprinted with permission from ref 19. Copyright 2003 Nature Publishing Group.

(water components are not included because they are identical to the solvent and do not contribute to the scattered intensity) and an average Rg of 45 nm (Figure 2, right). The fact of Rg = Rh is very intriguing. If the assemblies are solid spheres, Rg = 0.77Rh. For spherical objects, the only way of having Rg = Rh is to have all their mass distributed on their surface. This is a typical model for hollow spheres. Therefore, we can conclude that the {Mo154} assemblies form vesicle-like hollow, spherical structures in solution. The apparent Mw value, equivalent to ∼1150 {Mo154} macroions which can barely cover a single layer of a 45 nm sphere and the macroions are still not touching with each other, also confirms this. The accurate inter-{Mo154} distance is difficult to measure, but it is estimated to be 0.052 mM, or a certain amount of acetone is introduced into the solution, another distant peak appears (centered at ∼30 A˚ and extends the effective distribution to ∼34 A˚) in the p(r) plot (Figure 6, bottom). The original distribution remains unchanged, suggesting that the macroions still exist as discrete ions (the self-assembly process is slow; see below).32 This additional peak suggests that some counterions are closely associated with the macroions and distribute in the range of 0.2-0.9 nm to the surface of macroions. The peak due to the associated counterions becomes more and more significant with increasing POM concentration or acetone content. Meanwhile, Gunier plots indicate that the average Rg value of the {Mo72V30} macroions also increases accordingly. The 9206 DOI: 10.1021/la902917q

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appearance of the peak due to associated counterions is consistent with the appearance of the blackberry structures, indicating the direct connection between these two issues and the role of counterions on the blackberry formation. Compared to nanosized POM macroions, the large blackberry structures are much more powerful in attracting counterions. As estimated from zeta potential analysis, in aqueous solution, >99% of the effective charge on the blackberries has been neutralized by the closely associated counterions.23 A simple theoretical approach based on a mean-field model confirms the above experimental results regarding the interaction between two macroions in salt-free solutions with medium dielectric constant ε, with the aim to address the attraction of two like-charge macroions.33 All small counterions are categorized into two groups: free and bound cations.34 The formation of a bound cation is assumed due to strong electrostatic attraction with macroions, dictated by a free energy gain Δμbm relevant to the equilibrium ratio of bound and free cations. A chemical potential is incorporated to determine the chemical equilibrium between bound and free counterions, which is related to the interactions arising from (1) bound counterions and a macroion, (2) bound counterions and a second macroion, and (3) bound counterions and bound counterions (from both the original macroion and the second macroion).32 The chemical potential contributed from (1) is treated as an adjustable parameter in the model. The reduced interaction potential V(H) between two macroions is defined as the energy difference when their separation is decreased from infinity to the distance H, the shortest distance between the surfaces of the two spherical macroions with bound counterions. Figure 7, left shows the average number of bound counterions ÆNbæ with ε for various macroionic concentrations for H = 6 and 80 A˚. ÆNbæ decreases with increasingε or increasing macroionic concentration, qualitatively consistent with the experimental results. Figure 7, middle shows the V(H) values against ε for different macroionic concentrations for H = 6 A˚. For all concentrations, V(H) first decreases with decreasing ε and gradually exhibits attraction at low ε, suggesting that a low dielectric constant medium can enhance the attraction and then the self-assembly. After passing through the minimum, V(H) increases with decreasing ε and gradually approaches zero, suggesting that the self-association is less favored when further increasing the acetone concentration, consistent with experimental observations.25 For a higher macroionic concentration, the minimum V(H) value shifts to a larger ε, indicating that selfassociation can occur in a more polar solvent.

Role of Hydrogen Bonding Another important driving force for the blackberry formation is the hydrogen bonds formed between adjacent POMs on the blackberry surface. By using dielectric relaxation measurements, Oleinikova et al. noticed that, during the self-assembly of {Mo154} wheel-shaped macroions in aqueous solution, “the strength of the hydration extends as cluster aggregation takes place with more water molecules being more strongly bound between the wheels and the presence of relatively fewer less strongly bound water molecules”;35 that is, the water stays between {Mo154} macroions (33) (a) Lau, A. W. C. Phys. Rev. E 2008, 77, 011502. (b) Shew, C.-Y.; Yethiraj, A. J. Chem. Phys. 1997, 106, 5706. (c) Shew, C.-Y.; Yethiraj, A. J. Chem. Phys. 1999, 110, 11599. (34) (a) Manning, G. S. Ber. Bunsen-Ges. 1996, 100, 909 and references therein . (b) Liverpool, T. B.; M€uller-Nedebock, K. K. J. Phys.: Condens. Matter 2006, 18, L135. (35) Oleinikova, A.; Weing€artner, H.; Chaplin, M.; Diemann, E.; B€ogge, H.; M€uller, A. ChemPhysChem 2007, 8, 646.

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Figure 7. Plot of variation of the mean number of bound counterions ÆNbæ with dielectric constant ε when H = 6 A˚ (solid lines) and 80 A˚ (dotted lines) (left), and interaction potential V(H) against ε when H = 6 A˚ (middle) for different macroion concentrations from 10-6 to 10-4 M; (right) schematic plot denoting two macroions of radius a separated by a distance H surrounded by cations with bound cations confined between radii a and b. Reprinted with permission from ref 32. Copyright 2009 Wiley InterScience.

tions. This is a typical case for a charge regulated self-assembly process. Without clarifying the nature of the attractive forces, a general expression for the blackberry radius R is expressed as37 R ∼ -48λB u=ψ2

Figure 8. Plot of the average blackberry radius (in Rh) versus the inversed dielectric constant (1/ε) of the solvent for various POM macroions in water/acetone mixed solvents. Linear relationship roughly follows for these systems.

and shows higher viscosity (i.e., more hydrogen bonds formed). In other words, the additional hydrogen bonds help to “glue” the hydrophilic surface of POMs together. The special hydrogen bonding formed between macroions on the blackberry surface is reflected by the softness of the blackberry wall.36 An AFM study indicates that when placing {Mo72Fe30} blackberries on a silica surface, their height continues to drop with time while their overall morphology on the x-y plane (the surface) does not change. This can be explained as that the blackberries continue to lose their internal water when exposed on a surface. The strong hydrogen bonds prevent them from falling apart. In other words, the soft and robust blackberry surface makes them behave like “water balloons”.

Accurately Controlling the Blackberry Size Earlier in the text, it was demonstrated that the blackberry size can be controlled by adjusting the solvent content. A closer examination shows that the average blackberry size increases linearly with increasing 1/ε, with ε being the solvent’s dielectric constant. Examples for {Mo132},37 {Mo72V30},38a and {W72Mo60}38b in water/acetone mixed solvents are shown in Figure 8. Similar trends have also been identified in other POM macroionic solu(36) Liu, G.; Cai, Y.; Liu, T. J. Am. Chem. Soc. 2004, 126, 16690. (37) Verh€off, L.; Kistler, M. L.; Bhatt, A.; Pigga, J.; Gr€onewold, J.; Klokkenburg, M.; Veen, S.; Roy, S.; Liu, T.; Kegel, W. K. Phys. Rev. Lett. 2007, 99, 066104. (38) (a) Kistler, M. L.; Liu, T.; Gouzerh, P.; Todea, A. M.; M€uller, A. Dalton Trans. 2009, 26, 5094. (b) Sch€affer, A.; Merca, A.; B€ogge, H.; Todea, A. M.; Kistler, M. L.; Liu, T.; Thouvenot, R.; Gouzerh, P.; M€uller, A. Angew. Chem., Int. Ed. 2009, 48, 149.

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ð1Þ

with λB ∼56/ε. Consequently, the size of the blackberries is determined by three parameters: the solvent content (in ε), the effective charge on the blackberries (described by the zeta potential ψ), and the magnitude of the attractive force among the macroions (u, with its nature unidentified). At this moment, this expression is valid for the various POM (and other types of macroions) solutions we have explored. There are several ways of tuning the blackberry size besides adjusting the solvent content. A major approach is to adjust the charge density of macroions. Obviously, accurately tuning the charge density of POM macroions is also important for different applications, especially if considering the fact that the charge density of POMs often cannot be easily tuned to desired values from synthesis. For “weak acid” type POMs, tuning solution pH can nicely adjust the charge density on the macroions, and consequently the blackberry size can be tuned (see the following section). Another approach is to interact POM macroions with a few cationic surfactants to reduce their charge density. It requires a basic assumption that the long-tail cationic surfactants have to interact stoichiometrically with the macroions to ensure accurate control of the macroions’ charge density. A typical study was carried out with {Mo72V30}, which does not show self-assembly behavior in dilute aqueous solutions due to its high charge density.39 After introducing a small amount of water-soluble surfactants, such as cetyltrimethylammonium bromide (CTAB), trimethyltetradecylammonium chloride (CTAT), dodecyltrimethylammonium bromide (DTAB), and octyltrimethylammonium bromide (OTAB), the charge density on {Mo72V30} macroions is expected to decrease so that the {Mo72V30} macroions will enter the blackberry-formation regime (Figure 9). The long-chain CTAB and CTAT surfactants can interact with {Mo72V30} stoichiometrically. As a result, {Mo72V30} blackberries can be observed at the {Mo72V30} to surfactant molar ratio of 1:1.2, and the average Rh of the blackberries continues to increase with increasing surfactant concentration, as shown in Figure 10. The hydrophobic interaction from the surfactant tails might also contribute to the self-assembly, but the effect should be very minor as there are only a few alkyl chains on the relatively large surface area of each macroion. The above conclusions do not completely apply when interacting {Mo72V30} with less hydrophobic DTAB (with C12H25 alkyl chains). For 0.5 mg/mL {Mo72V30} aqueous solution, blackberry formation starts when ∼2.0  10-4 M DTAB (39) Kistler, M. L.; Patel, K. G.; Liu, T. Langmuir 2009, 25, 7328.

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Figure 9. Gradually introducing alkyltrimethylammonium halide cationic surfactants into the dilute aqueous solution of {Mo72V30} clusters can gradually decrease the charge density on the {Mo72 V30} macroions and induce blackberry formation. The average blackberry size increases with increasing surfactant amount (i.e., decreasing charge density on {Mo72V30}). Reprinted with permission from ref 39. Copyright 2009 American Chemical Society.

Interacting POM macroions with an excessive amount of surfactants will lead to the formation of hydrophobic, surfactantencapsulated POM clusters that can undergo different self-assembly and gelation processes, which have been extensively reported.40

Figure 10. Average hydrodynamic radius (Rh) of the {Mo72V30} blackberries in aqueous solution containing CTAB or CTAT, measured by DLS. The concentration of {Mo72V30} is 0.5 mg/mL, equivalent to a molar concentration of 2.6  10-5 M. Reprinted with permission from ref 39. Copyright 2009 American Chemical Society.

surfactant (i.e., at ∼1:4 molar ratio of {Mo72V30} to surfactant) is introduced, suggesting a higher critical association concentration (CAC) for the blackberry formation than those for CTAB and CTAT. It is an indication that DTAB surfactants do not interact stoichoimetrically with {Mo72V30} macroions; that is, there is still a considerable amount of single DTAB chains in solution. Over the CAC, in DTAB solution, the {Mo72V30} assembly size also increases with increasing surfactant concentration. However, the {Mo72V30}/DTAB blackberries are smaller in size than the other two at a given {Mo72V30}/surfactant ratio, suggesting higher effective charge density on the {Mo72V30} macroions. The short OTAB surfactant (contains two C8 alkyl chains) has the highest CAC value for blackberry formation and smallest blackberry size at a given {Mo72V30}/surfactant molar ratio. An interesting feature of the {Mo72V30}/OTAB solutions is that the average size of the {Mo72V30}/OTAB blackberries does not increase significantly with increasing OTAB concentration. Instead, blackberry size remains roughly constant at Rh ∼ 45 nm when the {Mo72V30}/OTAB molar ratio increases to up to 1:20. A reasonable explanation is that when more OTAB surfactants are introduced to the solution, most of them exist as discrete molecules and do not interact with {Mo72V30} macroions closely. 9208 DOI: 10.1021/la902917q

Weak Electrolyte Type POM Macroions Weak electrolyte type POM clusters, represented by Keplerates {Mo72Cr30}15 and {W72Fe30},13 are very unique. They exist as almost neutral clusters in single crystals and become macroanions in solution due to the partial deprotonation of their water ligands attached to the non-Mo (or W) centers. For example, {Mo72Fe30} has 30 potential deprotonation sites. When dissolved in pure water to make a 1.0 mg/mL solution, the solution pH is 3.4, that is, seven to eight protons are released from each {Mo72Fe30} cluster.38a,41 If assuming that the different deprotonation sites are independent of each other due to the relatively large distance (this is roughly valid when the overall degree of deprotonation is low) between them, the {Mo72Fe30} cluster can be treated as a weak acid with a single pKa value. Its degree of deprotonation and the charge density changes in response to solution pH can be adjusted by adding a small amount of NaOH or HCl solution. Figure 11 shows the blackberry formation in {Mo72Fe30} aqueous solution.41 At pH < 2.9, {Mo72Fe30} clusters are almost uncharged and stay as soluble molecules in solution. In solutions with pH > 2.9, {Mo72Fe30} clusters are more charged. Self-assembly occurs with the blackberry size decreasing with increasing pH, from Rh ∼ 50 nm at pH =3.0 to Rh ∼ 15 nm at pH = 6.0. The transition from single clusters to blackberries with the change in pH again confirms that van der Waals attractions are not the major attractive forces for the selfassembly. The transitions between blackberries with different pH can be achieved by adjusting solution pH.41 Scheme 1: 3þ 2þ þ III III 3 3 3 Fe ðOH2 Þ þ H2 OS 3 3 3 Fe ðOHÞ þ H3 O (40) (a) Kurth, D. G.; Lehmann, P.; Volkmer, D.; M€uller, A.; Schwahn, D. J. Chem. Soc., Dalton Trans. 2000, 3989. (b) Kurth, D. G.; Lehmann, P.; Volkmer, D.; C€olfen, H.; Koop, M. J.; M€uller, A.; Du Chesne, A. Chem.;Eur. J. 2000, 385. (c) Volkmer, D.; Bredenkotter, B.; Tellenboker, J.; K€ogerler, P.; Kurth, D. G.; Lehmann, P.; Schnablegger, H.; Schwahn, D.; Piepenbrink, M.; Krebs, B. J. Am. Chem. Soc. 2002, 124, 10489. (d) Li, W.; Bu, W.; Li, H; Wu, L.; Li, M. Chem Commun. 2005, 3785. (e) Bu, W.; Li, H.; Li, W.; Wu, L.; Zhai, C.; Wu, Y. J Phys. Chem. B 2004, 108, 12776. (f) Li, H.; Qi, W.; Li, W.; Sun, H.; Bu, W.; Wu, L. Adv. Mater. 2005, 17, 2688. (41) (a) Liu, T.; Imber, B.; Diemann, E.; Liu, G.; Cokleski, K.; Li, H.; Chen, Z.; M€uller, A. J. Am. Chem. Soc. 2006, 128, 15914. (b) Liu, T. J. Am. Chem. Soc. 2002, 124, 10942.

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Figure 11. Average hydrodynamic radii (Rh) of the blackberries formed in 0.5 mg/mL aqueous solutions of {Mo72Fe30} at different pH (adjusted by NaOH or HCl), as measured by DLS at 90 scattering angle. TEM images of aggregates on carbon film formed at pH ∼ 3.0 (left; conventional TEM) and pH ∼ 4.6 (right; more appropriate cryo-TEM). Reprinted with permission from ref 41a. Copyright 2006 American Chemical Society.

Figure 12. (left) Three-dimensional structure of a single Pd6L4 metal-organic nanocage molecule form supramolecular structures determined by SLS, DLS, and TEM techniques. (right) TEM studies of 0.06 mg/mL nanocage in water/acetone mixed solvent containing 40 vol % acetone: (A) 38-40 nm radius assemblies, (B) enlarged image, and (C) comprehensive view of burst and intact assemblies which show the soft, membranelike property. Reprinted with permission from ref 45. Copyright 2008 American Chemical Society.

{Mo72Cr30} clusters behave very similarly to {Mo72Fe30} in aqueous solution. Its pKa value is slightly lower; that is, at a given pH, they carry a little fewer charges.38a Consequently, much larger blackberries are formed in {Mo72Cr30} solution at a given pH. The size difference between {Mo72Cr30} and {Mo72Fe30} blackberries is significant and cannot be explained only by their charge density difference. A possible explanation is the difference between the lability of their surface water ligands. The water ligands attached to Cr3þ centers are very inert, resulting in more stable and stronger hydrogen bonds formed between {Mo72Cr30} macroions, which leads to larger blackberries.

Weak Acid Salt Type POM Macroions Between the strong electrolyte type and weak electrolyte type POM clusters, there exists a group of clusters which stay in the middle, and can be treated as weak acid salt type electrolytes (similar to the case of Na2HPO4). Represented by {Mo72V30}14 and {P4Y8W43},18 they carry a considerable amount of charges in crystals but still further deprotonate some surface water ligands when dissolving in a solvent. For these macroions, their selfassembly processes can be controlled by either changing solvent content or changing solution pH (in aqueous solution). Consequently, such POMs are valuable for directly comparing the effects of solvent content and solution pH on the blackberry size. The yttrium-containing lacunary polyoxotungstate {P4Y8W43} macroanions can form ∼53 nm blackberries in 0.3 mg/mL (42) Mishra, P. P.; Jing, J.; Francesconi, L. C.; Liu, T. Langmuir 2008, 24, 9308.

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aqueous solution.42 The blackberry size increases with increasing amount of acetone added into the solution, which is quantitatively similar to the case of {Mo132} as described earlier in the article.37 Changing solution pH can also alter the blackberry size. However, since the pH change only affects the amount of protons released from the macroions, the change of blackberry size due to pH is smaller than the change caused by changing solvent content.

Cationic Metal-Organic Nanocages While most POM clusters exist as anions in solution, there exists a large group of macrocations, represented by the metal-organic hybrid nanocages. Such cages are formed by coordinating metal ions with multiple organic ligands.43 Exploring the solutions of such nanocages might help to answer (1) if the macrocations have similar self-assembly behavior and (2) if other types of macroions besides POMs also demonstrate similar solution properties. A typical study was based on the commercially available (Wako) Pd6L4 nanocage [Pd = Pd(ethylenediamine), L = 2,4,6-tris(4-pyridyl)-triazine]44 (Figure 12), which has an octahedral shape and a diameter of ∼2 nm. Each nanocage molecule carries 12 positive charges owing to 6 PdII ions balanced by 12 NO3- counterions. Such cationic nanocages do not possess the homogeneous, hydrophilic surface of the large POM clusters described above. Instead, they contain multiple (43) Caulder, D. L; Raymond, K. N. Acc. Chem. Res. 1999, 32, 975. (44) Fujita, M.; Oguro, D.; Miyazawa, M.; Oka, H.; Yamaguchi, M.; Ogura, K. Nature 1995, 378, 469.

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Figure 13. (left) Change of total scattered intensity of {Mo72Fe30} solutions at 90 scattering angle. All solutions were kept at 25 C except

one at 45 C (the data shown by2). (right) CONTIN analysis of DLS study on {Mo72Fe30} aqueous solution at different times. Reprinted with permission from ref 46. Copyright 2003 American Chemical Society.

Figure 14. Possible mechanisms of {Mo72Fe30} blackberry formation in dilute aqueous solution. The upper mechanism has been proven to be correct based on SLS and DLS results, while the bottom mechanism can be ruled out. Adapted with permission from ref 23. Copyright 2006 American Chemical Society.

hydrophobic organic ligands so that they have both (separated) hydrophilic and hydrophilic domains on (near) their surface. In aqueous solution, Pd6L4 cationic nanocages exist as soluble discrete ions. When introducing acetone into the solution, they self-assemble into vesicle-like structures, as confirmed by DLS, SLS, and TEM studies.45 Because the nanocages contain separated hydrophobic domains, hydrophobic interaction should be considered for the self-assembly (i.e., the assemblies could be bilayer vesicles or blackberries). From the fact that the nanocages do not assemble in aqueous solution in which hydrophobic interaction is expected to be much stronger than in water/acetone mixed solvents, the hydrophobic interaction is unlikely to be the major driving force for the self-assembly; that is, the vesicle-like structures formed by nanocages are more likely also to be blackberry structures. Moreover, the size of these vesicles increases linearly with increasing 1/ε in water/acetone mixed solvents, suggesting a charge-regulated process similar to the self-assembly of POM macroions.45 Some other types of metal-organic nanocages also demonstrate similar solution behavior.

The Kinetic Properties of the Self-Assembly The slowness of the blackberry formation under certain circumstances enables detailed study on the mechanism of the selfassembly. Figure 13 demonstrates a typical SLS and DLS study on the formation of {Mo72Fe30} blackberries in aqueous solution at different macroionic concentrations.46 At room temperature, the self-assembly process takes months to reach equilibrium. The scattered intensity recorded by SLS continues to increase with time, suggesting the continuous formation of large structures in (45) Li, D.; Zhang, J.; Landskron, K.; Liu, T. J. Am. Chem. Soc. 2008, 130, 4226. (46) Liu, T. J. Am. Chem. Soc. 2003, 125, 312.

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Figure 15. Increment of the scattered intensity (I) from 0.5 mg/mL {Mo72Fe30}/H2O solutions at different temperatures (22, 35, 45, and 55 C) with time indicates the progress of blackberry formation. (inset) Calculation of the activation energy (Ea) for the blackberry formation. Reprinted with permission from ref 47. Copyright 2005 American Chemical Society.

solution. At the same time, the CONTIN analysis from DLS studies indicates that there exist two modes (corresponding to two different types of particles) in solution: one with Rh ∼ 1.2 nm which can be assigned to discrete {Mo72Fe30} macroions (unimers) and the other with average Rh ∼ 25 nm which should be attributed to large assemblies. The first peak becomes smaller and smaller while the second one grows larger with time, indicating that the discrete macroions are continuously forming blackberries. Interestingly, the average blackberry size remains almost unchanged during the whole process. Combining the above information, we conclude that the formation mechanism of the blackberry formation should follow route (b) in Figure 14.23 That is, at the beginning, the unimers slowly associate into dimers Langmuir 2010, 26(12), 9202–9213

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Figure 16. (left) Comparison of scattered intensity increment (A) and Rh change (B) of two {Mo72Fe30} samples along reaction time in 0.9 wt % NaCl and salt-free solutions Reprinted with permission from ref 48. Copyright 2009 American Chemical Society. (right) Light scattering study of the assembly of HPV capsid proteins at various HPV concentrations. The lag time, reaction slope, and extent of assembly were dependent upon the initial protein concentration. Changes in scattered light were not observed until minutes later. Reprinted from ref 52, with permission from Elsevier.

(or oligmers). This is the rate-determining step. Once enough oligmers are available, they quickly assemble into large blackberries. This step is fast so that no “small” blackberries are observed during the whole process. The slowness of the blackberry formation is attributed to the high energy barrier for the transition from single macroions to blackberries.47 Time-resolved SLS studies are used to determine the initial “reaction” rates in {Mo72Fe30} aqueous solutions at different temperatures. By using the Arrhenius equation ln(k) = -Ea/RT þ B, the activation energy of the oligmer formation can be calculated as ∼115 ( 8 kJ/mol, which is indeed very high (Figure 15).

Lag Phase during the Blackberry Formation: Connection to the Virus Capsid Formation Process In aqueous solution containing no or small amount of extra salts (e.g., NaCl, NaBr, NaI, and Na2SO4 at concentrations of 0.017 mol/L), the {Mo72Fe30} blackberry formation curves recorded by SLS studies are similar to that of a first-order reaction.47 However, a close look at the time-resolved SLS studies reveals that there is a short lag period at the beginning (in minutes; see Figures 4, 8, and 10 in ref 50). This lag phase becomes significant when the extra salt concentration is higher, as shown in Figure 16 (left), where NaCl (0.9 wt % or 0.17 mol/L) is introduced into a 10.0 mg/mL {Mo72Fe30} solution.48 After removing the minor precipitates due to the addition of salts, the remaining solution is a stable, saturated {Mo72Fe30}/NaCl solution (∼7 mg/mL) at room temperature. The initial scattered intensity from this solution is very low, suggesting that almost all the {Mo72Fe30} macroanions exist as discrete ions. After a lag period of almost 20 days, the intensity suddenly starts to increase until it is stabilized after months at a very high level, indicating a slow formation of large structures. Overall, the whole kinetic curve is sigmoidal with an extended lag phase. The sigmoidal curve for a self-assembly process has also been reported in other systems such as the self-assembly of virus capsid (47) Liu, G.; Liu, T. Langmuir 2005, 21, 2713. (48) Zhang, J.; Li, D.; Liu, G.; Glover, K. J.; Liu, T. J. Am. Chem. Soc. 2009, 131, 15152. (49) Prevelige, P. E.; Thomas, D.; King, J. Biophys. J. 1993, 64, 824. (50) Zlotnick, A.; Johnson, J. M.; Wingfield, P. W.; Stahl, S. J.; Endres, D. Biochemistry 1999, 38, 14644. (51) Ceres, P.; Zlotnick, A. Biochemistry 2002, 41, 11525. (52) Casini, G. L.; Graham, D.; Heine, D.; Garcea, R. L.; Wu, D. T. Virology 2004, 325, 320. (53) Bachmann, P. A.; Luisi, P. L.; Lang, J. Nature 1992, 357, 57. (54) Coveney, P. V.; Nemerton, A.; Boghosian, B. M. J. Am. Chem. Soc. 1996, 118, 10719.

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Figure 17. Continuous size distribution c(s) analysis of {Mo72Fe30} solution versus sedimentation coefficient, s. Experiments were performed at a {Mo72Fe30} concentration of 10 mg/mL in 170 mM NaCl solution at 20 C. Reprinted with permission from ref 48. Copyright 2009 American Chemical Society.

proteins (Figure 16, right),49-52 ester hydrolysis,53,54 vesicle formation,55 and nanoparticle preparation.56 In general, the sigmoidal curve is considered to be a typical feature of a two-step process: in the initial lag period, the “reaction” begins with the slow formation of oligomer nucleus; once the amount of the ratelimiting nucleus has reached a critical value, subsequent oligomers or monomers are quickly added to the growing assembly structures at a time until it is complete.57 A premise of the above discussions based on the assumption that the lag period shown in SLS studies is not due to the limited sensitivity of the instrument, which has been confirmed in the original article.48 To identify the oligomeric state during the lag period, sedimentation velocity (SV) experiments are performed on the 18th day after the sample solution is prepared, corresponding to the final stage of the lag period in the kinetic curve (Figure 16). In a typical SV experiment, raw data points are displayed as absorbance traces obtained from time-dependent changes in solution absorbance as a function of the radial position. The experimental curves were fitted using the Lamm equation to deduce the sedimentation coefficients (s) of sedimentating species and (55) Veronese, A.; Luisi, P. L. J. Am. Chem. Soc. 1998, 120, 2663. (56) Song, Y.; Yang, Y.; Medforth, C. J.; Pereira, E.; Singh, A. K.; Xu, H.; Jiang, Y.; Brinker, C. J.; von Swol, F.; Shelnutt, J. A. J. Am. Chem. Soc. 2004, 126, 635. (57) Hofrichter, J.; Ross, P. D.; Eaton, W. A. Proc. Natl. Acad. Sci. U.S.A. 1974, 71, 4864.

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Figure 18. (left) Formation of fluorophore-containing {Mo72Fe30} blackberries in solution. The additional cations, once added into solution, instantly interact with fluorophores in bulk solution and on blackberry surfaces, subsequently enter into the blackberries, and interact with the fluorophores inside. The anions could not cross the membrane. (right) Change in fluorescence quantum yield of Coumarin 1, 6-MQ, and CTC with addition of KBr, KCl, and CaCl2, respectively; (a) instantaneous change occurs with the addition of salts; (b) change in fluorescence quantum yield with time, once the addition of salt is stopped. Reprinted with permission from ref 60. Copyright 2008 American Chemical Society.

their corresponding sedimentation coefficient distributions c(s) (Figure 17). For the {Mo72Fe30} solution on the 18th day, the results show the coexistence of two species: s ∼ 6.6 S with the dominant abundance (56%) and s ∼ 9.5 S (10%).48 The theoretical sedimentation coefficients for {Mo72Fe30} monomers and oligomers are 6.9 S for {Mo72Fe30} monomers, 11.0 S for dimers, and 14.5 S for trimers, if assuming all the species are spherical. Therefore, the species with s ∼ 6.6 S corresponds to the monomers in solution (this is confirmed by studying the freshly prepared {Mo72Fe30} solution in which only monomers with s ∼ 6.6 S are identified). The species with s ∼ 9.5 S is attributed to dimers. This value is lower than the theoretical value, possibly due to the fact that the dimers are not spherical. In addition, the measurement also suggests the possible existence of a small amount of larger oligomers (such as trimers) in solution. However, the very small amount is not sufficient to lead to reliable results from such studies. Besides the concentration of additional salts, the length of the lag period also depends on temperature, the valent state of the cations and the anions, as well as the solvent content.48 Importantly, the assembly structures and formation processes of blackberries and virus capsids (mostly spherical, single-layered structures formed by the ordered assembly of capsid protein units which are also soluble macroanions) demonstrate interesting similarities.49-51 For example, the in vitro assembly process of human papillomavirus (HPV) from protein subunits to icosahedral HPV capsids displays a sigmoidal kinetic curve in SLS studies (Figure 16, right). The lag period before the rapid growth of assemblies is dependent on the protein concentration and the ionic strength. The theoretical nucleation-elongation model perfectly explains the delay time and suggests that HPV assembly begins with the slow formation of dimers of protein subunits.52 Similar results were also reported in the in vitro assembly process of hepatitis B virus capsids49,58 and cowpea chlorotic mottle virus capsids.59 In addition, the assembly of virus capsid proteins is also sensitive to temperature and ionic strength. Considering that both virus capsid proteins and POMs are nanoscale, soluble macroions and both self-assemble into singlelayered spherical structures, it is reasonable to postulate that their self-assemblies might share similar mechanisms and maybe even (58) Zlotnick, A. Virology 2003, 315, 269. (59) Zlotnick, A.; Aldrich, R.; Johnson, J. M.; Ceres, P.; Young, M. Virology 2000, 277, 450.

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driving forces. The hydrophobic interaction is widely believed as the dominant driving force for the virus capsid formation. However, for the POMs, they do not contain any hydrophobic moieties, and thus, hydrophobic interaction does not play a role for the blackberry formation. Then the question is: is it possible that the electrostatic interaction might be underestimated in the virus shell formation? Ideally and potentially, the POM macroions might be useful as simple model systems to study the more complicated biomacromolecular systems.

Permeability of Blackberry “Membrane” to Small Cations The blackberry “membranes”, although formed by pure inorganic compounds, are impressively soft and robust. They are different in nature from cell membranes because there is no closely packed hydrophobic region in the middle. For cell membranes, it is difficult for small cations to transport over freely, and special ion channels are needed in order to achieve biological functions. Formed by macroanions which are not in touch with each other, the membranes might be permeable to cationic species. Fluorescence spectroscopy is sensitive to examine the transport of small ions through the blackberry membrane. The watersoluble dyes specifically sensitive for one (or two) type of ions, such as chlorotetracycline (CTC) for Ca2þ and Mg2þ, 6-methoxyquinoline (6-MQ) for Cl- and Coumarin 1 for Br-, are introduced into freshly prepared {Mo72Fe30} aqueous solution which does not contain any blackberries yet. The dye molecules will be partially incorporated into the blackberries during the selfassembly, as confirmed by an 18 nm shift of their fluorescent signals. This shift also shows that the environment inside the blackberries is different from the bulk solution, as the water inside might have a higher viscosity due to the existence of a larger amount of hydrogen bonds formed close to {Mo72Fe30} macroions. Time-resolved fluorescence anisotropy results further confirm the coincidence between the change of the incorporation of dyes and the blackberry formation.60 After the completion of blackberry formation, additional salts containing the type of ions sensitive to the specific dye are introduced into the solution (here we use CTC and Ca2þ as an example). A sudden jump in fluorescence signal suggests that the CTC molecules staying in bulk solution are immediately saturated (60) Mishra, P. P.; Pigga, J.; Liu, T. J. Am. Chem. Soc. 2008, 130, 1548.

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by the dominant amount of Ca2þ ions introduced. However, after this initial process, a very slow, continuous increment in the fluorescence signal appears, suggesting a slow, continuous binding between Ca2þ ions and CTCs (Figure 18). This can be explained as that the Ca2þ ions slowly transport over the blackberry membranes and are able to bind with the CTC molecules inside. This is strong evidence to show that small cations are able to move across the blackberry membrane. Experiments by using Mg2þ cations lead to the same results and conclusions. On the contrary, small anions such as Cl- and Br- cannot pass through the blackberry membrane. This is reasonable since the blackberry membrane is negatively charged and should repel with those small anions.60

contain some hydrophobic parts), demonstrate intriguing behavior in dilute solutions which is different from that of simple ions and large colloidal suspensions. The macroions tend to selfassemble into hollow, spherical, “blackberry” structures with their size being tunable with changing solvent content, charge density of macroions, additional salts, and sometimes solution pH. The counterion-mediated attraction and hydrogen bonds are expected to be the driving forces for the self-assembly. The blackberry structure, as well as its formation mechanism, mimics the formation of virus shells by virus capsid proteins. The blackberries show the feature of biomembranes, and their membranes are permeable to small cations.

Conclusions Hydrophilic macroions, mainly represented by the polyoxometalates (POMs) and metal-organic hybrid molecules (which

Acknowledgment. T.L. acknowledges support of this work by the National Science Foundation (CHE0545983), the American Chemical Society (PRF 46294-G3), the Alfred P. Sloan Foundation and Lehigh University.

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