Diversiform and Transformable Glyco-Nanostructures Constructed from AmphiphilicSupramolecular Metallocarbohydratesthrough Hierarchical Self-Assembly: TheBalance between Metallacycles andSaccharidesGuang Yang,†,¶,∇ Wei Zheng,‡,∇ Guoqing Tao,† Libin Wu,† Qi-Feng Zhou,† Zdravko Kochovski,⊥

Tan Ji,‡ Huaijun Chen,† Xiaopeng Li,∥ Yan Lu,⊥,# Hong-ming Ding,*,§ Hai-Bo Yang,*,‡

Guosong Chen,*,† and Ming Jiang†

†The State Key Laboratory of Molecular Engineering of Polymers and Department of Macromolecular Science, Fudan University,Shanghai 200433, PR China‡Shanghai Key Laboratory of Green Chemistry and Chemical Processes, School of Chemistry and Molecular Engineering, EastChina Normal University, Shanghai 200062, PR China§Center for Soft Condensed Matter Physics and Interdisciplinary Research, School of Physical Science and Technology, SoochowUniversity, Suzhou 215006, PR China∥Department of Chemistry, University of South Florida, Tampa, Florida 33620, United States⊥Soft Matter and Functional Materials, Helmholtz-Zentrum Berlin fur Materialien und Energie, 14109 Berlin, Germany#Institute of Chemistry, University of Potsdam, 14467 Potsdam, Germany¶Biomass Molecular Engineering Center, Anhui Agricultural University, Hefei, Anhui 230036, PR China

*S Supporting Information

ABSTRACT: During the past decade, self-assembly of saccharide-containing amphiphilic molecules toward bioinspired functionalglycomaterials has attracted continuous attention due to their variousapplications in fundamental and practical areas. However, it stillremains a great challenge to prepare hierarchical glycoassemblieswith controllable and diversiform structures because of thecomplexity of saccharide structures and carbohydrate-carbohydrateinteractions. Herein, through hierarchical self-assembly of modulatedamphiphilic supramolecular metallocarbohydrates, we successfullyprepared various well-defined glyco-nanostructures in aqueous solution, including vesicles, solid spheres, and openedvesicles depending on the molecular structures of metallocarbohydrates. More attractively, these glyco-nanostructures canfurther transform into other morphological structures in aqueous solutions such as worm-like micelles, tubules, and eventupanvirus-like vesicles (TVVs). It is worth mentioning that distinctive anisotropic structures including the openedvesicles (OVs) and TVVs were rarely reported in glycobased nano-objects. This intriguing diversity was mainly controlledby the subtle structural trade-off of the two major components of the amphiphiles, i.e., the saccharides and metallacycles.To further understand this precise structural control, molecular simulations provided deep physical insights on themorphology evolution and balancing of the contributions from saccharides and metallacycles. Moreover, the multivalencyof glyco-nanostructures with different shapes and sizes was demonstrated by agglutination with a diversity of sugar-binding protein receptors such as the plant lectins Concanavalin A (ConA). This modular synthesis strategy provides

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Received: September 9, 2019Accepted: October 25, 2019Published: October 25, 2019

Artic

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© 2019 American Chemical Society 13474 DOI: 10.1021/acsnano.9b07134ACS Nano 2019, 13, 13474−13485

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access to systematic tuning of molecular structure and self-assembled architecture, which undoubtedly will broaden our horizonson the controllable fabrication of biomimetic glycomaterials such as biological membranes and supramolecular lectin inhibitors.

KEYWORDS: glycomaterials, diversiform structures, hierarchical self-assembly, metallocarbohydrates, anisotropic structures

In biological systems, biomolecules such as proteins,phospholipids, and DNA, etc., hierarchically self-assembleinto diversiform, regular, and functional supramolecular

structures, such as actin fibers, microtubules, vesicle-like cellmembranes, and various viruses,1−4 at nano- or micrometerscale, via multiple dynamic noncovalent interactions. Anenhanced understanding of the formation mechanisms ofthese various supramolecular assemblies with specific functionswill bring us not only the understanding of basic biologicalprocesses in detail,5,6 but also the construction of bioinspiredfunctional materials for different practical applications.7 Thus, agreat deal of effort on the design and construction of well-defined hierarchical supramolecular nanoarchitectures frombiomacromolecules has been witnessed during the past twodecades.8−10 For example, DNA, peptides and proteins havebeen extensively explored as biomacromolecular building blocksfor developing diverse hierarchical supramolecular nanostruc-tures with different functionalities.11−14 These structures notonly cover different dimensions, i.e., from one-dimensional (1D)nanowires, 2D nanosheets, to 3D crystalline frameworks, butalso feature high anisotropic varieties with precise control.Carbohydrates, which are a predominant class of biomole-

cules, play crucial roles in different biological processes, such ascell recognition, cell adhesion, and signal transfer, which makethem attractive building blocks for constructing functionalmaterials with potential applications in medical, pharmaceutical,and environmental fields.15−20 While comparing to nucleic acidsand peptides as building blocks for supramolecular architectures,carbohydrates are far behind because of their complex structuresand hardly controllable noncovalent interactions, although theyhave great abundancy and functionalities on cell surfaces.21 Tomimic the natural glycocalyx on cell surface and also to constructfunctional materials, various glycopolymers, glycopeptides, andglycoamphiphiles have been utilized for the construction ofintriguing self-assembled glyco-nanostructures, such as spheres,fibers, and tubes in nanometer and micrometer scale.22−29 Forexample, Lee and co-workers reported the construction ofvarious carbohydrate-coated assemblies (micelles, vesicles, andcylinders) from sugar-modified amphiphilic rods (tetra-(phenylene) and oligo(ethylene oxide) substituted withmannose), via slightly varying the length of oligo(ethyleneoxide) and the numbers of tetra(phenylene) rod.30 Percec et al.fundamentally studied the self-assembly of amphiphilic Janusglycodendrimers and reported a series of glycodendrimersomeswith different shape and size depending upon the molecularstructures.31,32 Although much progress has been made in thefield of carbohydrate-based self-assembly in aqueous media, it isstill a great challenge to obtain diversiform morphologies andparticularly to realize their precise control by changing thecarbohydrate structures. To the best of our knowledge,interesting anisotropic morphologies, including opened vesicles(OVs) and tupanvirus-like33 vesicles (TVVs), with vase-likeshape based on saccharide-containing building blocks have notbeen realized so far. It is probably caused by the difficulties incontrolling the precise structures of saccharide-containingpolymers and the noncovalent interactions between carbohy-drates, including multiple hydrogen bonds and van der Waals

interactions,34 which are quite complicated and hardly tuned incomparison to the noncovalent interactions between DNA orpeptides.Recently, discrete metallacycles formed through coordina-

tion-driven self-assembly have been proved to be promisingscaffolds for nanometer-sized aggregates with well-defined sizeand shape via hierarchical self-assembly.35,36 Inspired by thedistinctive properties of the carbohydrates, currently, two casesof construction of carbohydrate-based discrete metallacycleshave been reported by Stang and co-workers.37,38 Likewise,considering the great diversity, stability, and directionality ofmetal−ligand interactions, we envisioned that saccharide-containing amphiphilic metallacycles are endowed with precisecontrol on the saccharide structures, including conjugationpositions, conjugation numbers, thus making them elegantcandidates for the construction of various glyco-nanostructureswith diversiform and controllable morphologies. Meanwhile, itshould be noted that the topological change of metallacyclesprovides an opportunity of tuning the shape and size of thesaccharide-containing supramolecular amphiphiles.Herein, we report the hierarchical self-assembly of amphi-

philic supramolecular metallocarbohydrates in aqueous sol-ution, where saccharide and metallacycle have been demon-strated as two individual components controlling the self-assembled morphology coordinately. Via precisely tuning thenumber of saccharides and the metallacycle scaffolds, micelles,vesicles, and opened vesicles (OVs) were observed with well-controlled size and distribution. More importantly, thesemorphologies can further transform into more complexstructures such as tubules, worm-like micelles, and tupanvirus-like vesicles. Some anisotropic morphologies, including the OVsand tupanvirus-like structures, which are rarely observed inliteratures were successfully achieved. Dissipative particledynamics (DPD) and Brownian dynamics (BD) simulationswere employed to demonstrate the contributions of metalla-cycles and saccharides to the significant structural diversity ofthe obtained glyco-nanostructures and the energy-favorablefusion way of OVs, respectively. Finally, the saccharide moietieson the surface of these nano-objects demonstrated theirbiological functions via different agglutination kinetics withmodel lectins controlled by different morphologies.

RESULTS AND DISCUSSIONDesign, Synthesis, and Characterization of Metal-

locarbohydrates. According to the general principle of“directional bonding” and the “symmetry interaction”, thediscrete supramolecular coordination complexes with well-defined shape and size can be prepared by the reaction of thespecific building blocks with different angles and symmetry.39−47

In this study, four saccharide-containing donor building blockswith different angles D1-Man (0°) (Man: monosaccharidemannoside), D1-Mal (0°) (Mal: disaccharide maltoside), D2-Man (120°), andD2-Mal (120°) can be combined with suitableacceptors to induce self-assembly of metallocarbohydrates withprecise control on carbohydrate modifications. The 0° or 120°saccharide-containing dipyridyl donors were synthesizedthrough several simple steps starting from the commercially

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available 3,5-dibromophenol such as palladium-catalyzedSonogashira reaction, esterification reaction and thiol−enereaction (Supporting Information). With the aim to constructthe multiple metallocarbohydrates with different sizes, two typesof di-Pt(II) acceptors A1 (180°) and A2 (120°) with differentangles were employed, respectively. Stirring a mixture of 0°donors D1-Man or D1-Mal with 180° di-Pt(II) acceptor A1 inMeOH at room temperature for 8 h in a 1:1 ratio, led to theformation of self-assemblies 2-Man or 2-Mal, respectively.According to the same assembly process, the hexagonalmetallacycles 3-Man, 3-Mal, 6-Man, and 6-Mal were preparedwith 120° donors D2-Man or D2-Mal combined with anequimolar amount of a 180° or 120° acceptor A1 and A2,respectively (Scheme 1). Multinuclear magnetic resonance (1HNMR and 31P NMR) results indicate the formation ofsupramolecular metallocarbohydrates with certain highlysymmetric structures. As shown in Figure 1a−e, the 31P{1H}spectra of 2-Man, 3-Man, and 6-Man all showed a sharp singletat 13.7−13.9 ppm with concomitant 195Pt satellites (ca. 13.97ppm for 2-Man, 13.73 ppm for 3-Man, and 13.70 ppm for 6-Man), which exhibited an obvious upfield shift compared withthose of relevant starting diplatinum(II) acceptors A1 (ca. 22.04ppm) and A2 (ca. 22.11 ppm). This change as well as thereducing of the coupling of the flank 195Pt satellites (ca. ΔJ =−139.7 Hz for 2-Man, ΔJ = −176.2 Hz for 3-Man, and ΔJ =−194.5 Hz for 6-Man), is corresponding to the electron back-donation from the platinum species. In the 1H NMR spectrum(Figure S1) of each metallocarbohydrates, signals correspond-

ing to the Hα and Hβ of the pyridine rings exhibited obviousdownfield shifts caused by the loss of electron cloud density.These clear-cut signals of both the 31P{1H} and 1HNMR spectraalong with the good solubility of these species imply theformation of a discrete structure as the most dominatedassembly product. Formation of 2-Mal, 3-Mal, and 6-Mal wasalso confirmed via the same methods (Figure S2 and S3).In order to further confirm the molecular compositions of

multicharged supramolecular structures, electrospray ionizationtime-of-flight mass spectrometry (ESI-TOF-MS) was utilized todetermine the stoichiometry of the assembly structure, becausethis method is often capable of remaining the metallacyclesintact furthest during the ionization process. In the mass spectraof 2-Man, the three main peaks at m/z = 1691.99, 1079.04, and771.78, agree well with the diverse charge state caused by theloss of counterions [M − 2OTf]2+, [M − 3OTf]3+, and [M −4OTf]4+ (Figure 1f), respectively, in which M denotes the intactsupramolecular metallocarbohydrate assembly. These observedexperimental isotope patterns of peaks were in good agreementwith the corresponding theoretic calculated results. Briefly, themass data successfully prove the formation of 2-Man. Similarly,the observed peaks of 3-Man and 6-Man inmass were also nicelyconsistent with the corresponding theoretic isotope patterns(Figure 1g,h). Notably, the higher noise of the elemental isotopepatterns of peaks of 6-Man was possibly due to partiallydisassociation of the supramolecular structures during the ESI-TOF-MS test. These results of ESI-TOF-MS spectrometry gavefurther support for the formation of the designed metal-

Scheme 1. Self-Assembly of Acceptors and Saccharide-Containing Donors To Form Hexagonal Metallocarbohydrates 2-Man, 3-Man, 6-Man, 2-Mal, 3-Mal, and 6-Mal

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locarbohydrates. Because of the difficulties of growing singlecrystals of metallocarbohydrates, we turned to take advantage ofthe PM6 semiempirical molecular orbital simulation for thegeometrical optimizations of 2-Man, 3-Man, and 6-Man. Asexpected, the optimized architecture of three saccharide-functionalized metallacycles all exhibited very similar, androughly planar hexagonal rings as the core with saccharideslocating at the corners (Figure S4). In addition, the simulationalso displayed the internal and external radius for 2-Man being0.7 and 1.6 nm respectively, and for 3-Man 1.4 and 2.4 nm, andfor 6-Man, 2.5 and 3.4 nm, respectively.Multilayered Vesicles (MLVs) Formed from 2-Man.

When 2-Man was dispersed into water, the aqueous solutionturned to be opalescent (Figure S5), indicating the formation ofnano-objects. 1H NMR spectrum (Figure S6) of 2-Man in D2Oshowed the disappearance of proton signals from aromatic rings,implying the hydrophobic ring backbone was embedded in theassembled structure. Transmission electron microscopy (TEM)was utilized to observe the assemblies of 2-Man in water. Asshown in Figure 2a, spherical structures with an obvious lighter

contrast of the interior and wall were observed, which wasindicative of the vesicle structures. The average diameter wasabout 380 nm (calculated from 100 vesicles), which wasconsistent with the result from dynamic light scattering (DLS)(Figure 2b). It was notable that the average thickness of thevesicles is about 65 nm (Figure S7), implying that they are themultilayered vesicles (MLVs) as the external diameter of 2-Manwas ∼3.2 nm. Cryogenic-TEM (Cryo-TEM) was furtheroperated to confirm the MLV structure (Figure 2c and FigureS8). More strikingly, TEM image at a larger magnificationrevealed one single-layered vesicle with thickness ∼3.2 nm closeto the external diameter of 2-Man, located inside the MLV(Figure 2a inset image). This was also confirmed by Cryo-TEM(one single-layered vesicle with diameter ∼140 nm; thickness∼3.4 nmwas inside amultilayered vesicle) (Figure 2c and FigureS8), which agrees well with the TEM findings. Moreover,scanning TEM (STEM) also confirmed such structures, and thecorresponding elemental mapping via energy dispersive X-ray(EDX) spectroscopy just supported the outside MLVs withuniform distribution of the compositional elements on the wallof vesicles (Figure 2d,e). Possibly, the resolution of EDX is nothigh enough to observe the internal single-layered vesicle. Inshort, a combination of the techniques fully supported thesuccessful formation of intriguing glyco-nanostructures ofMLVsfrom the hierarchical self-assembly of hexagonal 2-Man (Figure2f).To provide physical insights into the self-assembled MLVs by

2-Man in the experiment, dissipative particle dynamics (DPD)simulation (see details of the DPD method in SupportingInformation) was employed to display the time evolution of theself-assembly process at the microscopic/mesoscopic level. Themodeling of 2-Man was established by using the coarse-grained(CG) method, where each group in the metallocarbohydrateswas represented by one CG bead in the simulation (see the insetof Figure 2g and Figure S9). As shown in Figure 2g, hundreds of2-Man molecules were first randomly placed in the solvatedsimulation box. Since the main part of the metallacycles washydrophobic, the molecules quickly formed small aggregates. Astime went on, the small aggregates further formed into the largeaggregates. Moreover, due to the π−π stacking (i.e., effectivelyattractive interaction between aromatic beads), the largeaggregates gradually turned into regular and layered structureswith metallacycle inside and carbohydrates outside. Moreinterestingly, these layered structures would contact with eachother due to the carbohydrate-carbohydrate interactions(CCIs),48−51 and finally became the multilayered structure. Ingeneral, the simulation results indicated that the multilayeredstructure was indeed one possible packing structure and stableform (of 2-Man assembly) due to the cooperation of the π−πstacking and CCIs, which was consistent to the observed resultsin the experiments.

Structures Formed by 3-Man and 6-Man. Interestingly,when 3-Man and 6-Man were dispersed in water, entirelydifferent self-assembly behaviors from that of 2-Man wereobserved under the same condition. The 1H NMR spectra(Figure S10 and S11) of assembled 3-Man and 6-Man in D2Oindicated similar assemblymechanism to that of 2-Man owing tothe disappearance of proton signals from aromatic rings.However, in the case of 3-Man, TEM images displayed thatthey self-assembled into solid spherical structures with anaverage diameter about 13 nm (Figure 3a and Figure S12),which agrees well with the DLS experiments (Dh ∼ 14 nm, PDI:0.243) (Figure 3b). Cryo-TEM (Figure 3c) and tapping mode

Figure 1. 31P NMR spectra (161.9 MHz, in CD3OD, 25 °C) of (a)180° acceptor A1, (b) 120° acceptor A2, (c) self-assembled hexagon2-Man, (d) self-assembled hexagon 3-Man, and (e) self-assembledhexagon 6-Man. Theoretical (blue) and experimental (red) ESI-TOF-MS spectra of (f) 2-Man, (g) 3-Man, and (h) 6-Man.

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atomic force microscope (AFM) (Figure 3d) both revealed thesimilar spherical morphologies with a narrow size distribution.To understand the distinct self-assembly behavior of 3-Man,DPD simulation was again employed. Similar to 2-Man, the 3-Man molecules also first formed small aggregates due to thehydrophobic interactions. However, since the size of metalla-cycles of 3-Man was larger than that of 2-Man, the moleculescould not pack as regularly as 2-Man did, not resulting in thelayered structures. The exposed hydrophobic parts tend toaggregate into small micelles with carbohydrates on their outersurfaces, which was in good agreement with the experimentalfindings. Notably, some of the micelles fused into elongatedmicelles due to CCIs, which was also observed in theexperiments (see the red box in Figure 3c).In the case of 6-Man, intriguingly, TEM experiment revealed

that it formed OV structures with a diameter around 160 nm(Figure 3f,g and S13). This unusual morphology was furtherconfirmed by Cryo-TEM observations (Figure 3h and S14),indicating that the opening existed inherently on the vesicles inaqueous solution. DPD simulation also showed that the finalassembly was curved and irregular structure (Figure S15). Asshown in Figure S15, due to the large size of metallacycle andmultiple interacting sites in 6-Man, the molecules initiallyformed cross-linked network. Then the network shrank to the

layered structure to avoid the undesired interactions betweenthe hydrophobic parts and water. However, the packing of the 6-Manmolecules was not regular; thus, the layered structure herewas irregular and far from the ordered multilayer found from 2-Man assembly, which may give some hints to the OV structuresobserved in the experiment. Moreover, STEM as well as thecorresponding EDX spectroscopy elemental mapping (FigureS16) were also performed and confirmed the compositionalelements. It is of great interest to note that two thin layers of theoxygen element located on internal and external wall of the OVstructure were found, which implied that carbohydrates are richon the internal and external surfaces, agreeing well with the 1HNMR results (Figure S11). Additionally, the distance betweenthe two thin oxygen layers was about 21 nm, which was in goodagreement with the thickness of OVs measured by TEM.

Morphology Transformation of Assembled Manno-side-Modified Metallacycles. The three metallocarbohy-drates showed different tendency in their hierarchical assemblyprocess. And considering the intrinsic dynamic nature of thecoordinated metallacycle structures and the carbohydrate-carbohydrate interactions between the initial assemblies, wespeculated that the initial morphologies were possibly kinetically“frozen” structures driven by the hydrophobic and π−πinteractions of the metallacycles. Thus, we further explored

Figure 2. (a) TEM image of 2-Man in aqueous solution. Inset: TEM image of a single vesicle at a higher magnification. (b) DLS result of 2-Manin aqueous solution. (c) Cryo-TEM, (d) STEM, and (e) the corresponding elemental mapping images of 2-Man. Scale bar: 250 nm.Representative internal single-layered vesicles were pointed by white rows. (f) The illustration of the assembly process of 2-Man in aqueoussolution. (g) Time sequence of snapshots illustrating the self-assembly process of 2-Man in the simulation.

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the possible morphology reshape of the assembled structuresduring long-standing time.Taking 3-Man as an example, after standing in solution over

the period of 5 weeks, TEM images revealed the coexistence ofspheres and fused short worm-like micelles (Figure 4a),indicating very slow fusion of the spheres to worm-likestructures. Then TEM and AFM were employed to trace thetransformation process with different time intervals. As shown inFigure 4 and Figure S17, the initial solid spheres stepwiselytransformed to short worm-like spheres, then longer worm-likespheres with branches, and finally complex network over as longas 15 weeks. This transformation process in water as a functionof time was also monitored by DLS (Figure S18). The long timefor morphological transformation was possibly due to the weakCCIs including hydrogen bonding and van der Waalsinteractions. In order to study the mechanism of this fusionprocess, Fourier-transform infrared spectroscopy (FT-IR) of theinitial spheres and final powdered samples of worm-likestructure were compared (Figure S19). It was found that theO−H stretching peak of initial spheres at 3448 cm−1 shifted tolower wavenumber 3438 cm−1 in the worm-like structures,which implies the formation of stronger hydrogen bonds in the

latter.50,52,53 Similar studies on the assemblies of 2-Man in waterwith standing time presented that theMLVs fused into multiple-walled tubes in ∼8 weeks (Figure S20a and 20b), along axial

Figure 3. (a) TEM image and (b)DLS result of 3-Man in aqueous solution. (c) Cryo-TEM image of 3-Man in aqueous solution (Red box showedthe small worm-like micelles). (d) AFM image of 3-Man in aqueous solution. (e) Time sequence of snapshots illustrating the self-assemblyprocess of 3-Man in the simulation. (f) TEM image of 6-Man in aqueous solution; inset: an enlarged TEM image on the opened vesicle. Theopen sites were pointed by white arrows. (g) Diameter distribution of 6-Man measured from TEM images. (h) Cryo-TEM image of openedvesicles with open sites pointed by white arrows.

Figure 4. TEM images of 3-Man in aqueous solution with standingtime increasing: (a) Short worm-like fibers after 5 weeks.Representative short fused fibers were shown in white boxes. (b)Branched fibers after 10 weeks. The branched junctions werepointed by white arrows. (c) Networks formed after 15 weeks. (d)Morphology transformation scheme of 3-Man in aqueous solutionwith standing time increasing.

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growth based on the coexistence of vesicles and tubules (FigureS21). It is worth mentioning that 31P{1H} spectra confirmed thestability of metallacycles after several months in water, viacollecting 2-Man, 3-Man, and 6-Man assemblies in waterthrough freeze-drying (Figure S22). The morphology trans-formation of OVs of 6-Man in water upon time seemed veryattractive. As shown in Figure S23, DLS results as a function oftime evidently showed that Dh of the assemblies increasedapparently, and after about 3 months,Dh reached about 260 nm.Surprisingly, the traced observations by TEM (Figure S24)displayed fusion of two OVs, into stable, uniform asymmetricvase-like structure, which is very similar to that of tupanvirus,33

thus called tupanvirus-like vesicles (TVVs) (Figure 5a,b). Itshould be noted that the TVVs were stable in water even afterstanding in solution for more than 7months (Figure S25). As weknow that this kind of asymmetric particles are very attractiveowing to their possible applications in different fields includingdrug delivery, artificial cell construction and catalysis, etc.,54−56

endowed by the subtle combination of hollow structures and thedistinguishing asymmetric architecture. It should be noted thatthere has been no report of glyco-nanostructures with similarmorphology obtained from saccharide-containing molecules

yet. Then, TVVs structures were further confirmed by SEM,Cryo-TEM, and STEM. As shown in Figure S26, SEM imageshowed high uniformity of the obtained TVVs, and the enlargedSEM image clearly displayed the open end of the TVVs in Figure5c. Cryo-TEM image showed the round-bottom TVVs with anopening at the end of the neck and a diameter of body size∼200nm (Figure 5d and S27).Moreover, STEM (Figure 5e) as well asthe corresponding EDX spectroscopy elemental mapping(Figure 5f−j) not only further revealed the structure of TVVs,but also confirmed the uniform distribution of the compositionalelements on the wall of TVVs, which fully supported that theirformation was from the hierarchical self-assembly of hexagonal6-Man. Furthermore, we noticed that the elemental mapsdisplayed similar distributions of most of the elements, like thecase of 2-Man. However, the diameter observed from oxygenwas about 162 nm, 10 nm thicker than those measured for theothers, i.e., 152 nm. This very interesting result indicated that athin layer (∼5 nm) of oxygen homogeneously surrounding theout surface of TVVs, which implied the assemblies were mainlycovered by carbohydrates on the out surface. Furtherinvestigation of transformation process of 6-Man provided usa deeper understanding of the self-assembly. Figure S24b and

Figure 5. (a,b) TEM, (c) SEM, and (d) Cryo-TEM images of TVVs formed in the solution of 6-Man after standing over 3 months. (e) STEMimage and (f−j) the corresponding elemental mapping images of the tupanvirus-like structure. Scale bar: 200 nm. (k) Schematic illustration ofthe CG models for opened vesicles in BD simulation. Three possible equilibrium states of two opened vesicles in the simulations (l) and thesystem energy (m) in the above three cases. (n) The occurring probability of the above three cases in the free assembly of eight vesicles duringthe simulation. (o) Morphology transformation scheme of 6-Man in aqueous solution with standing time increasing.

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S24c showed a TEM image of dimerization of OVs andcoexistence of the OVs and TVVs after incubation for 1.5, and 2months, respectively, which implied that the TVVs were formedby fusion of the OVs. This transformation process may beaccelerated by other treatments including heating (Figure S28).Considering 6-Man as an amphiphilic molecule, the above resultcould be even more interesting since the transformation fromOVs to TVVs indicates a more or less transition from kineticproduct to thermodynamic product. Such long incubation timeindicates a relatively high barrier for interconversion, which isquite different from common glyco-nanostructures made bysmall molecules. In literature, such high barrier and slow timescale were only observed in amphiphilic block copolymers.To reveal the physical mechanism underlying the formation of

TVVs in the experiment, Brownian dynamics (BD) simulations(see details of BDmethod in SI) were applied to study the fusionprocess of OVs at the mesoscopic level. For the sake ofsimplicity, here the OV was treated as the hollow sphere particlewith open sites (Figure 5k). Typically, there were three possibleways for the fusion of particles (Figure 5l), i.e., mouth-to-end (I),mouth-to-mouth (II), and end-to-end (III). However, due todifferent contact ways (line-to-surface contact in case I, line-to-line contact in case II, and point-to-point contact in case III), theenergy in these three cases was different (Figure 5m). Since thecontact area between the two particles in the mouth-to-end casewas the largest (Figure S29), the system energy in this case wasthe lowest among the three cases. Moreover, we also performedadditional simulations for the free assembly of eight particles(see SI Movie S1 BD simulation). Interestingly, the occurringprobability of mouth-to-end case in the simulations was also thelargest (Figure 5n). Collectively, from both thermostatic andkinetic view, we confirmed that case I was the optimal fusionway, which facilitated the transformation of OVs to TVVs(Figure 5o).Comparison of Nanostructures Formed byMan-Based

and Mal-Based Metallacycles. Man-based metallacycles (2-Man, 3-Man, and 6-Man) and Mal-based ones (2-Mal, 3-Mal,and 6-Mal) share the identical metallacycles but differentcarbohydrates, i.e., having monosaccharide (Man) and dis-accharide (Mal) respectively. We found that the two groupsshowed very different self-assembly behaviors under the samecondition. TEM, AFM, and STEM images all revealed that for 2-Mal, fibers with micrometer-scale length were obtained (Figure6a,b and S30), differing apparently from the vesicles formed by2-Man. It is noteworthy that either the diameters from TEM orheights from AFM showed nonuniform distributions, whichimplied that the 2-Mal fibers do not show the typical feature oflinear supramolecular polymers.57,58 In fact, the only differenceof 2-Man and 2-Mal came from the side carbohydrates, i.e.,monosaccharide against disaccharide. To present a possibleexplanation, DPD simulations were employed. As shown inFigure 6c, 2-Malmolecules first assembled into small aggregatesdue to their hydrophobic properties. Then, after the rearrange-ment and packing of these small aggregates via effective π−πinteractions, the large aggregates and layered structures formed,which was similar to the case of 2-Man. However, differing fromthat of 2-Man, the layered structures of 2-Mal would not befurther enlarged. Instead, they preferred to form the long layer-by-layer structures due to the stronger CCIs of disaccharide Malthan that of monosaccharide Man by four times (Figure 6d,e),making the assembly growth along the CCIs direction. For 3-Mal and 6-Mal, DLS results revealed their assembly with Dh ofabout several hundred nanometers (Figure S31 and S32). TEM

images displayed that both of them self-assembled into vesicleswith size about 200 nm (Figure 6f and h). Further, STEM as wellas the corresponding EDX spectroscopy elemental mappingdisplayed the vesicle structures formed by 3-Mal (Figure S33)and 6-Mal (Figure S34) in water, and also confirmed thecompositional elements with uniform distribution on the wall ofvesicles. Notice that the hydrophobicity of the moleculesdecreased with the increase of carbohydrate number, which may

Figure 6. (a) TEM image of 2-Mal in aqueous solution. (b) AFMimage of 2-Mal, inset is the height of the fiber at different sites. (c)Time sequence of snapshots illustrating the self-assembly process of2-Mal in the simulation. Time evolution of interaction energy(including CCI and π−π stacking) during the self-assembly processin the simulation: (d) 2-Man, (e) 2-Mal. (f) TEM image of 3-Mal inaqueous solution. Inset: An enlarged vesicle of TEM. (g) The finalsnapshot of self-assembled structure of 3-Mal in the simulation (top:side view; bottom: section view; the cyan beads stand for the water).(h) TEM image of 6-Mal in aqueous solution. Inset: An enlargedTEM image.

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facilitate the structure change from the micelle to the vesicle.59

In our simulations, vesicles formed by 3-Mal molecules wereobserved (Figure 6g). Meanwhile, the increase of carbohydratenumber also means the increase of CCIs. This could furtherincrease the contacts between molecules within assembledvesicles, which may be beneficial for the closure of these vesicles.Protein Recognition of Glyco-Nanostructures. As

mentioned above, the naturally ubiquitous and significantspecific recognitions between proteins and carbohydrates,which played vital roles in bioactive events,60,61 inspired us tofurther study the protein-binding ability of the series ofhierarchical self-assembled glyco-nanostructures from thehexagonal saccharide-functionalized metallacycles. We canenvision that the obtained solid spheres, MLVs and OVs, werecoated with carbohydrates due to their hydrophilicity, asindicated by the previously discussed 1H NMR (Figure S6,S10, and S11). As shown in Figure S35, glyco-nanostructures ofmetallacycles are stable upon dilution with HEPES buffer,indicating the nanostructures will be intact during proteinbinding experiment. The experiment was performed in HEPESbuffer (pH = 7.2) in the presence of 1 mMCa2+ and 1 mMMn2+

to make the Ca2+-dependent ConA binding possible. Theconcentration of mannoside of different glycostructures waskept constant, as 0.2 mM. Basically, the carbohydrate−proteininteractions can bemeasured by different techniques. A turbiditytest was widely used to monitor the cross-linking of glyco-nanostructures via protein-carbohydrate binding.31,62,63 Asshown in Figure 7, the time profile change of absorption spectra

at 450 nm displayed an increase of turbidity (with sameconcentration 0.2 mM of mannoside) after the addition ofConA, as a result of aggregation upon incubation. Notably, theturbidity increase of MLVs of 2-Manwas much lower than thoseof solid spheres (3-Man) and OVs (6-Man). This phenomenonwas supported by DLS, since during titration of ConA to theglycostructure solution, Dh of 2-Man remained almost constant,while those of 3-Man and 6-Man increased dramatically. TEMalso revealed the same phenomenon (Figure S36). However, byisothermal titration calorimetry (ITC) (Figure S37), almostsimilar heat release to the same amount of ConA was detectedfrom the three solutions, indicating that a similar amount ofbinding took place during the process. From the above results,we proposed that in the case of solid spheres of 3-Man and OVsof 6-Man, the protein-carbohydrate binding mainly took placebetween different glyco-nanoparticles, leading to significant

cross-linking of these structures. In the case of MLVs of 2-Man,the bindingmainly took place on the surface of the same vesicles,which does not result in cross-linking of vesicles. This isprobably because that the larger size and thickness of MLVseasily enable Con A (Dh∼ 8 nm) to adhere onto the surface andhardly to cross-link others. This result is quite important fordesigning glycoassemblies for future study, such as mimickingcell−cell interactions.

CONCLUSIONS

In summary, we successfully constructed a series of amphiphilicmetallocarbohydrates with precise and tunable structures byemploying coordination-driven self-assembly. Furthermore,such metallocarbohydrates were found to self-assemble intodiverse glyco-nanostructures, including multilayered vesiclecontaining single-layered vesicle, micelles, vesicles, OVs, andfibers, etc., in water, mainly depending on the structures ofmetallacycles and saccharides. Among these structures, the vase-like morphology seems evenmore attractive than the others withmany possibilities including further manipulation to hybridmaterials based on the anisotropic shape. With the support ofDPD simulation, the contributions from metallacycles andsaccharides were described separately, showing the subtle trade-off of these two components during self-assembly. Unexpect-edly, upon standing time increasing, the OVs dimerized forminguniform and anisotropic TVVs, where the transformationmechanism was revealed by BD simulation. To the best of ourknowledge, there have been few reports for these kinds ofanisotropic glyco-nanomaterials. The bioactivity of these well-defined glycomaterials was demonstrated via binding withConA.In this paper, the polymorphism of saccharides has reached an

amazing level by integrating with metallacycles; thus, program-mable nanostructures, especially anisotropic structures withbiological functions based on saccharides, could be designed andcontrolled. The successful marriage of metallacycle andsaccharide exhibits a bright future of the approach of hierarchicalself-assembly to design and develop well-defined and control-lable glycomaterials. Given the key roles of carbohydrates in life,it will inspire further studies on the hierarchical self-assembly ofsupramolecular saccharide-containing metallacycles, for poten-tial biological applications including drug/gene delivery,immunotherapy, and regulation of cell recognition.

METHODSCharacterization. NMR spectra were collected from AVANCE III

HD 400MHz from Bruker BioSpin International. 31P NMR resonancesare referenced to an internal standard sample of 85% H3PO4 (δ 0.0).Coupling constants (J) are denoted in Hz and chemical shifts (δ) inppm. Multiplicities are denoted as follows: s = singlet, d = doublet, m =multiplet. Matrix-assisted laser desorption ionization−time-of-flight(Maldi-TOF)Mass Spectra were collected from AB SCIEX 5800. Massspectra were collected from Bruker Compact ESI-Q-TOF-HRMS.TEM micrographs were acquired from a series of magnifications by aTecnai G2 20 TWIN (FEI), performed at 200 kV. The samples forTEM measurement were prepared by dropping the solution onto acarbon-coated copper grid. The carbon films on copper grids weretreated for hydrophilicity by glow-discharge before use. Thepreparation of TEM samples was as follows: 3 μL solution wasdropped onto the hydrophilic treated carbon film. After standing about1 min, the liquid was removed by filter paper. DLS was recorded by aZetasizer Nano ZS90 from Malvern Instruments (UK). All of the SEMimages were obtained using from Ultra 55 of Zeiss. The SEM sampleswere prepared on clean copper substrates. AFM images were obtained

Figure 7. (a) Agglutination assays of Man-containing glyco-nanoparticles (0.2 mM) and natural ConA (0.25 mg/mL) inHEPES buffer with incubation time up to 30 min. The inset cartonsrepresent the mixture state after an addition of ConA into differentglycoparticles in aqueous solutions. (b) Time-dependent sizechange of mixtures of 2-Man/ConA (red line), 3-Man/ConA(blue line) and 6-Man/ConA (black line).

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on a multimode 8 (Bruker), using Scanasyst mode. AFM samples wereprepared via depositing 5 μL solution on the freshly cleaved mica.Samples solution was standing on mica surface ∼2 min, and then 200μL water was used to wash the mica surface carefully. Then the washedsamples were dried in air. Cryo-TEMmicrographs were acquired from aseries of magnifications under 200 kV by JEOL JEM-2100. For thepreparation of Cryo-TEM samples, 4 μL solution was first droppedonto the hydrophilic treated carbon film. After standing about 30 s, theliquid was removed by filter paper, and then the samples were quicklyimmersed into the liquid ethane frozen by liquid nitrogen. ITCexperiments were operated on a MicroCal VP-ITC system at 20.00 ±0.01 °C. UV−vis spectra were recorded in a conventional quartz cell(light path 1 mm) on a UV-2550 spectrophotometer from Shimadzu,Japan. The turbidity experiments were operated by mixing ConA andassemblies at in HEPES buffer with the final concentration of ConA0.25 mg/mL and mannoside 0.2 mM on metallacycles.Representative Synthetic Procedures of Ligands and Metal-

locarbohydrates. (The full details of synthesis and characterization ofnew compounds were described in Supporting Informations.)Synthesis of D1-Man. 0.2 g (0.5 mmol) a5, 0.12 g (0.5 mmol) a3,

and 7 mg dimethylphenylphosphine were dissolved into 10 mL DMF.After stirring for 24 h at 50 °C under Ar protection, the solvent wasremoved by evaporation. The compound was separated viachromatography column using methanol/DCM mixture(Vmethanol:VDCM = 1:6) as eluting solvent. The collected solid productwas 27 mg. 1H NMR (400MHz, CD3OD, 298 K, Figure S44) δ 8.76 (t,2H); 8.57−8.55 (m, 2H), 8.06−8.03 (m, 2H), 7.69−7.68 (t, 1H);7.52−7.49 (m, 2H); 7.41−7.40 (d, 2H); 4.83 (d, 1H); 4.00−3.93 (m,1H); 3.85−3.80 (m, 2H); 3.75−3.69 (m, 3H); 3.64−3.51 (m, 3H);3.12−2.99 (m, 2H); 2.91−2.84 (m, 2H); 1.43−1.41 (d, 3H). 13CNMR(100 MHz, CD3OD, 298 K, Figure S45) δ 173.44, 173.42 151.22,150.91, 148.27, 139.35, 131.75, 125.23, 124.05, 123.70, 120.19, 100.31,90.29, 86.52, 73.46, 71.19, 70.73, 67.17, 65.74, 61.53, 40.32, 35.29,31.51. 29.30, 27.52, 24.51, 24.48. ESI-MS (m/z) [D1-Man + H]+

calculated for C32H33N2O8S 605.19, found 605.20 or [D1-Man + Na]+

calculated for C32H32N2O8SNa, 627.18, found 627.18 (Figure S46).Synthesis of 2-Man. The dipyridyl donor ligandD1-Man (6.04 mg,

1 μmol) and the organoplatinum 180° acceptor A1 (12.36 mg, 1 μmol)were weighed accurately into a glass vial. The vial was added 2.0 mLmethanol and the reaction solution was then stirred at roomtemperature for 4 h to yield a homogeneous light solution. Solidproduct 2-Man was obtained by removing the solvent under a vacuum.1HNMR (400MHz, CD3OD, 298 K, Figure S65) δ 9.03 (s, 2H), 8.84−8.86 (d, 2H), 8.25−8.82 (d, 2H), 7.76−7.84 (m, 3H), 7.52−7.54 (m,4H),); 4.83 (d, 1H); 4.00−3.93 (m, 1H); 3.85−3.80 (m, 2H); 3.75−3.69 (m, 3H); 3.64−3.51 (m, 3H); 3.12−2.99 (m, 2H); 2.91−2.84 (m,2H); 1.43−1.41 (d, 3H); 1.21−1.43 (m, 24H), 1.14−1.21 (m, 36H).31P NMR (161.9 MHz, CD3OD, 298 K, Figure S66) δ 13.97 (s, JPt−P =2677.4 Hz). ESI-TOF-MS of 2-Man calcd for [M − 2OTf]2+ 1690.96,found 1691.04; calcd for [M − 3OTf]3+ 1076.99, found 1077.05; calcdfor [M − 4OTf]4+ 770.75, found 770.78.Preparation of Assembly. The solid product of 20 mg 2-Man was

dissolved into 200 μLmethanol. Then, under mild stirring, 10mLwaterwas dropwise added into the solution within 30 min. The assemblyprocedures of 3-Man, 6-Man, 2-Mal, 3-Mal, and 6-Mal were similar.Molecular Simulation. The dissipative particle dynamics (DPD)

simulations were applied to investigate the underlying mechanism ofthe self-assembly behaviors of different metallacarbohydrates in water.The Brownian dynamics (BD) simulations were used to investigate thefusion mechanism of open vesicles (OVs). More details on thesimulation method are given in section v of SI.

ASSOCIATED CONTENT

*S Supporting InformationThe Supporting Information is available free of charge on theACS Publications website at DOI: 10.1021/acsnano.9b07134.

Movie S1 (MP4)

Materials, Details of the synthesis, characterizations ofligands and saccharide-functionalized metallacycles, fur-ther details of molecular simulation parameters, addi-tional TEM, STEM and elemental mapping, Cryo-TEM,SEM and AMF images, 1H NMR and FT-IR, DLS, andITC spectra (PDF)

AUTHOR INFORMATIONCorresponding Authors*E-mail: dinghm@suda.edu.cn.*E-mail: hbyang@chem.ecnu.edu.cn.*E-mail: guosong@fudan.edu.cn.ORCIDXiaopeng Li: 0000-0001-9655-9551Hong-ming Ding: 0000-0002-9224-4779Hai-Bo Yang: 0000-0003-4926-1618Guosong Chen: 0000-0001-7089-911XAuthor Contributions∇G.Y. and W.Z. contributed equally.NotesThe authors declare no competing financial interest.

ACKNOWLEDGMENTSG.C. thanks NSFC/China (No. 51721002, 21861132012,21975047, and 91527305). H.-B.Y. acknowledges NSFC/China (No. 21625202), STCSM (No. 16XD1401000), andProgram for Changjiang Scholars and Innovative ResearchTeam in University for financial support. G.Y. acknowledges thefinancial support of CPSF (No. 2017M621354 and2018T110335). We would also like to thank the Joint Lab forStructural Research at the Integrative Research Institute for theSciences (IRIS Adlershof, Berlin) for Cryo-TEM imaging. Thiswork was also funded by Open Research Fund Program ofShandong Provincial Key Laboratory of Glycoscience &Glycotechnology (Ocean University of China).

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