Self-Organized Mesostructured Hollow Carbon Nanoparticles via a

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Chemistry of Materials

Self-organized Mesostructured Hollow Carbon Nanoparticles via a Surfactant-free Sequential Heterogeneous Nucleation Pathway Hongwei Zhang, Meihua Yu, Hao Song, Owen Noonan, Jun Zhang, Yannan Yang, Liang Zhou and Chengzhong Yu* Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, QLD 4072, Australia KEYWORDS: Electron tomography, colloidal carbon, sol-gel process, invaginated morphology, heterogeneous nucleation ABSTRACT: Mesostructured hollow carbon nanoparticles have widespread applications. A big challenge in materials science is surfactant-free synthesis of hollow carbon nanoparticles with tunable mesostructures. Herein we report a new surfactant-free sequential heterogeneous nucleation pathway to prepare mesostructured hollow carbon nanoparticles. This strategy relies on two polymerizable systems, i.e. resorcinol-formaldehyde and tetraethyl orthosilicate, each of which undergoes homogeneous nucleation and particle growth. By controlling the polymerization kinetics of two systems when mixed together, sequential heterogeneous nucleation can be programmed leading to monodispersed and mesostructured hollow carbon nanoparticles with large mesopores, controllable mesostructures (bi- and triple-layered) and rich morphologies (invaginated, intact and endo-invaginated spheres). For the first time, it is demonstrated that the invaginated structure shows better hemocompatibility compared to the intact one. The pristine hollow carbon nanoparticles with large pore size and high pore volume show high loading capacity of biomolecules and successfully deliver biomolecules into cells. Our strategy has paved the way for the designed synthesis of unprecedented carbon nanostructures with potential applications in drug/biomolecule delivery.

Introduction Sol-gel derived monodispersed nanoparticles, formed via the homogeneous nucleation and growth of a precipitating phase from solution, have provided an extensive array of functional materials for advanced applications.1-3 Monodispersed nanoparticles with various compositions, including silica, metal oxides and polymers, have attracted tremendous attention due to their simple preparation methods, tunable size, ease of modification, intriguing physicochemical properties, and remarkable utility as templates and building blocks.4-12 Among them, carbon nanospheres including hollow and mesoporous structures have recently been extensively studied for applications in biology, environment and energy, because these types of carbon materials possess high surface area, large pore volume, hydrophobicity, chemical and thermal stability, electrical conductivity, and biocompatibility.13-16 Up to date, several strategies have been developed to synthesize nanostructured carbon spheres, including chemical vapor deposition (CVD), nanocasting with preformed nanoparticles, and soft-templating assisted synthesis.17-23 The CVD or nanocasting strategies are usually complicated and time consuming. The size, morphologies and pore structures of the replicated carbon materials are highly limited by their parent templates. Soft-templating

assisted synthesis with phenol formaldehyde resin as the carbon precursor is an effective strategy to fabricate mesostructured carbon nanospheres with controllable pore structure and particles size.24-28 However, the mesostructures of carbon nanospheres can only be achieved by introducing surfactants to the synthesis system. Previous attempts to simultaneously polymerize tetraethyl orthosilicate (TEOS), resorcinol and formaldehyde (RF) in a one-pot synthesis led to hollow carbon spheres with only microporous walls.29 It remains challenging to synthesize mesostructured hollow carbon spheres in the absence of surfactants. Moreover, control of morphological symmetry of mesostructured hollow carbon spheres and, more importantly, its impact on bio-applications has not been reported. Herein, we report a new sequential heterogeneous nucleation (SHN) pathway to prepare self-organized carbon nanospheres with controllable mesostructures and morphologies in the absence of surfactants. The SHN approach is schematically illustrated in Figure S1 (see also details in Experimental section). The synthesis is carried out in an EtOH/water system with NH3·H2O as the catalyst, simply using TEOS, resorcinol and formaldehyde as precursors. When TEOS and RF precursors are added (step I), Stöber spheres form first through a homogenous

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Figure 1. Characterization of invaginated and intact MHCS. SEM (a, c) and TEM (b, d) images of invaginated and intact MHCS, respectively. Digital images (e) of two MHCS dispersed in aqueous solutions showing the Tyndall effect and the particle size distribution curves (f) by DLS measurement. Scale bars are 200 nm.

nucleation process due to the relatively faster condensation rate compared to the RF system. Once the silica spheres are formed, the RF precursors preferentially condense on the silica surface through heterogeneous nucleation. In order to tune the wall structure, TEOS is introduced for the second time at a chosen time point (step II), which leads to the formation of uniformly distributed silica nanoparticles on the RF shell through a subsequent heterogeneous nucleation process. The residual RF oligomers co-condense with silica nanoparticles to form a composite RF/silica layer. After carbonization (step III) and silica removal (step IV), mesostructured hollow carbon spheres (MHCS) with a bilayered structure are obtained. By controlling the thickness of carbon shell through hydrothermal treatment or the thickness of silica interlayer through tuning the TEOS amount in step II, the bilayered morphology can be finely regulated from intact spheres to invaginated or endo-invaginated spheres. The invaginated morphology demonstrates advantages over the intact one in terms of loading capacity of biomolecules and hemocompatibility, providing a great potential in intravascular applications of biomolecules. Experimental Section Synthesis of MHCS Monodispersed SiO2@RF@SiO2@RF composites was synthesized via a one-pot sol-gel process under alkaline condition in alcohol-water system. In a typical synthesis,

tetraethyl orthosilicate (TEOS, 2.8mL), resorcinol (0.4 g) and formalin (37 wt %, 0.56 mL) were added to the solution composed of ammonia aqueous solution (3.0 mL, 28 wt %) , deionized water (10 mL) and ethanol (70 mL). The mixture was vigorously stirred for 6 h at room temperature followed by the addition of 1.5 mL of TEOS and kept stirring for 24 h. The mixture was further hydrothermal treated at 100 °C for 24 h in a Teflon-lined autoclave. Both the solid products (without hydrothermal treatment and with hydrothermal treatment) were collected by centrifugation and dried at 50 °C for overnight. Finally, MHCS were harvested after carbonization at 700 °C under N2 atmosphere and removal of silica templates by hydrofluoric acid (HF, 5 %) etching. The other products prepared with different amounts of TEOS added in the second step are denoted as MHCS-x and MHCS-xHT, where x represents the TEOS amount (mL) and HT means the samples are hydrothermal treated before carbonization. Materials characterizations The morphology and structure of the samples were investigated by field emission scanning electron microscope (FESEM, JEOL 7800) operated at 2 kV and transmission electron microscope (TEM, JEOL 2100) at 200 kV. Nitrogen adsorption isotherms were measured at 77 K using a TriStar II Surface Area and Porosity analyzer (Micromeritics). The samples were degassed under vacuum for 6 hours at 180 °C before analysis. Dynamic light scattering


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(DLS) measurements were carried out at 25 °C on a Malvern Zetasizer Nano ZS Instrument. X-ray photoelectron spectra (XPS) were collected on a Kratos Axis ULTRA Xray photoelectron spectrometer (Perkin-Elmer). X-ray diffraction (XRD) patterns were performed on Bruker D8 Advanced X-Ray Diffractometer with Co Kα radiation (λ=0.179 nm). The ET specimens were prepared by dispersing the samples in ethanol followed by ultrasonication and then depositing the suspension directly onto copper grids (2000×1000 slot, Proscitech) with Formvar supporting films. Colloidal gold particles (10 nm) were deposited on both surfaces of the grid as fiducial markers for the subsequent image alignment procedures. The tomographic tilt series were carried out by tilting the specimen inside the microscope around double axis from +70° to -70° at an increment of 1° under the electron beam. Alignment and 3D reconstructions of MHCS was carried out on IMOD software. For the time-investigated RF samples for cryo-TEM, one drop of the suspension at various time points was withdrawn and deposited on to the copper grid. The excess liquid on the cooper grid was removed by filter paper. Subsequently, the copper grip was immersed into liquid nitrogen in a small centrifuge tube and freeze dried to remove all the liquid in a freeze dryer. Then the samples were investigated by transmission electron microscope. Results and Discussion From the scanning electron microscopy (SEM) images (Figure 1a), MHCS prepared without hydrothermal treatment in step III exhibit an invaginated spheroidal morphology, much like a deflated balloon where one side of the sphere enfolds towards the other. Transmission electron microscopy (TEM) images of the invaginated MHCS show a clearly bilayered and hollow internal structure (Figure 1b). When MHCS are prepared with hydrothermal treatment, an intact spheroidal morphology is obtained as shown by the SEM image (Figure 1c). TEM observations for these particles also show a bilayered concentric spherical structure (Figure 1d). Invaginated and intact MHCS exhibit uniform outer diameters of 250 and 270 nm, respectively (measured from both SEM and TEM images). Both particles disperse well in aqueous solution and produce the characteristic Tyndall effect commonly observed for colloidal suspensions (Figure 1e). Dynamic light scattering (DLS) measurements reveal a hydrated particle size of 265 and 295 nm for invaginated and intact MHCS, respectively (Figure 1f). The narrow size distributions and low polydispersity index (PDI of 0.1) for two samples indicate both MHCS possess highly uniform particle size and excellent water dispersibility. High resolution SEM images reveal highly porous external surfaces with open-pore entrances for the invaginated MHCS. Intact MHCS, on the other hand, exhibit relatively smooth and continuous surface morphology (Figure S2). For three-dimensional (3D) nano-objects with complex internal structures such as MHCS, investigation by conventional TEM may provide misleading information. This

is because TEM images provide the collective structural information over a certain thickness and merge it into a 2D projection. For example, the fine structures between the inner and outer layers are not clear. Moreover, it seems that two spheres shown in Figure 1b (indicated by arrows) are not invaginated, although this effect could result from the electron beam passing perpendicular to the plane of invagination. Electron tomography (ET) is a rapidly developing technique for the advanced 3D imaging of complex structures, which allows virtual reconstruction of a material’s internal structure using 3D models built from a series of 2D slices.30, 31 We used the ET technique to study the detailed structures of MHCS. A series of tilted images were taken in the range of +70 to -70° with an increment of 1°. From the Supplementary Movie S1, one can clearly observe the invaginated MHCS particle apparently changing from an invaginated to an intact spherical structure. This highlights the ambiguity of the data provided by conventional TEM alone and confirms the importance of ET characterization for materials with complex and asymmetrical architectures. To observe the detailed internal structures of MHCS, electron tomograms were generated from two perpendicular tilting series using IMOD software.32 The ET slice which cuts perpendicular to the invagination face of the MHCS (Figure 2a) exhibits a clearly bilayered, crescent moon-like morphology. The inner and outer layers are linked by thin carbon bridges of approximately 1−2 nm in thickness (indicated by black arrows). In contrast, a tilt-series of the intact MHCS reveals a complete spherical morphology throughout the rotation (data not shown). The ET slide in Figure 2b shows a full moon-like morphology for the intact MHCS, where the two concentric

Figure 2. Electron tomography (ET) characterizations of invaginated, intact and endo-invaginated MHCS. ET slides of invaginated MHCS (a) and intact MHCS (b), TEM image (c) and ET slide (d) of endo-invaginated MHCS. Scale bars are 100 nm.



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layers are linked by more substantial carbon bridges with approximately 4−5 nm in thickness. The invaginated and intact samples also differ noticeably in thickness and the degree of continuity of inner and outer shells. The outer layers of the invaginated and intact samples appear relatively continuous with an average thickness of 6 and 12 nm, respectively; however, the inner layer of the invaginated structure shows numerous defects and interruptions which form a more fragile and discontinuous inner shell when compared to the intact structure. The average interlayer spaces are measured to be approximately 15 and 20 nm for the invaginated and intact MHCS, respectively. Digitally reconstructed structures for two MHCS particles are shown in Figure S3. The orange and yellow colors represent the inner and outer shells, respectively. Invagination of both the inner and outer shells can be observed for the invaginated MHCS, while spherical morphology is seen for the intact MHCS, which is consistent with the morphological observations from TEM and SEM. Moreover, carbon bridges linking the inner and outer shell can also be observed for both invaginated and intact MHCS. Nitrogen sorption studies for both invaginated and intact MHCS show type-IV adsorption isotherms (Figure S4). The BJH pore sizes calculated from the adsorption branch indicate pore sizes of 15.9 and 18.0 nm for the invaginated and intact MHCS, respectively. These pore sizes correspond closely with the interlayer distance between the inner and outer shells observed in ET and TEM micrographs, suggesting this confined interlayer space is responsible for the pore size distribution. The BET surface area and pore volume of invaginated MHCS (1032 m2 g-1 and 2.11 cm3 g-1, respectively) are slightly higher than those obtained for the intact MHCS (880 m2 g-1 and 1.44 cm3 g-1, respectively), which may be attributed to thinner shells. The X-ray photoelectron spectra (XPS) in Figure S5 show that only peaks from C1s (~285 eV) and O1s (~534 eV) are detected, revealing the major components of both invaginated and intact MHCS are carbon and oxygen.33 The mass percentage of carbon and oxygen are calculated to be 92.9 % and 7.1 %, respectively. The X-ray diffraction (XRD) patterns reveal the amorphous nature of MHCS (Figure S5). In order to understand the formation mechanism of MHCS, we systematically studied the nucleation and growth processes of silica-RF particles as a function of time. Since both TEOS and RF can independently polymerize under Stöber conditions to form uniform solid particles, their individual reaction kinetics were first investigated (Figure 3, curve I and II, Figures S6 and S7). Under our synthesis conditions, the polymerization of TEOS results in formation of silica particles within 15 minutes (m), consistent with the typical induction period commonly observed in Stöber sphere formation.34 These spheres then rapidly increase in size up to 2 h, after which particle size is relatively consistent. RF polymerization, on the other hand, forms spheres with slower growth. The

Figure 3. Statistical analysis of particle sizes as a function of reaction time. Pure silica (curve I), pure RF (curve II), silica@RF (curve III) and silica@RF@silica@RF (curve IV). The average particle sizes are calculated based on TEM measurements for more than 50 particles for each sample (mean ± s.d.).

formation of some irregular RF polymer nuclei is observable at 1 h, which continue developing into spherical particles by 2 h. The RF spheres increase in size relatively rapidly from 2 to 6 h followed by a slower growth until 12 h. When TEOS and RF are added simultaneously, the particle size initially (until 2 h) follows the same trend as the pure silica system (Figure 3 curve III, Figure S8). After 2 h, silica@RF core-shell structures begin to form and the thickness of RF shell increases as a function of time. No evidence of solid RF spheres can be found, indicating that the RF polymerization system has been changed from homogeneous to heterogeneous nucleation on the surface of silica cores, consistent with classical nucleation theory that the free energy barrier for heterogeneous nucleation on a surface is considerably lower as compared to homogeneous nucleation.35 However, this approach leads to hollow carbon spheres with only microporous walls,29 which has little control over the morphology and mesostructure of final products and thus limited applications. When TEOS is introduced in step II at a carefully chosen time point of 6 h, TEM was used to monitor the structural evolution over the following 2 h. From TEM images of samples after calcination in air (Figure S9), it can be seen that a secondary population of silica nuclei appear on the surface of silica@RF particles within 15 m after the second TEOS addition. The secondary silica nanoparticles increase in size from ~5 nm at 15 m up to ~10 nm at 30 m after which the silica nanoparticles merge together to form a relatively continuous interlinked shell with a thickness of 18 nm. After secondary TEOS addition, the particle size steadily increases (Figure 3, curve IV) relative to the silica@RF particles shown in curve III, achieving an additional 30 nm in diameter after 12 h of growth. TEM data confirm the absence of any solid silica nanoparticles in the final products. The above observations indicate that the RF layer of silica@RF particles formed in step I triggers a subsequent heterogeneous nucleation of TEOS.


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Due to the slow polymerization behavior of the RF system, the RF precursors remaining in solution preferentially nucleate on the surface of secondary silica nanoparticles. The sequential heterogeneous nucleation of two polymerizable systems and their interplay gives rise to an interpenetrating silica-RF composite shell structure. Removing silica after carbonization results in the final structures of MHCS. The ET results of fine structures of MHCS are in accordance with the SHN mechanism. The bridges in between two carbon layers come from the intergrowth of RF with secondary silica nanoparticles. Hydrothermal treatment favors further condensation of RF, leading to thicker bilayers as well as bridges and eventually intact MHCS. The invaginated MHCS with exposed porous surfaces are formed due to the thinner RF layers and bridges when hydrothermal treatment is not applied in step III. Our SHN mechanism can recur over additional nucleation cycles. As a demonstration, a third addition of TEOS and RF precursors was introduced into the system. The resulting triple-layered MHCS structures (Figure S10) are consistent with another cycle of heterogeneous nucleation. The added TEOS heterogeneously nucleates on the RF surface, forming an additional population of silica nanoparticles, followed by heterogeneous nucleation and growth of RF over silica. The use of SHN pathway under the same polymerization conditions for multiple cycles provides scope for the design of nanomaterials with elegant structures. Judicious selection of time points for the addition of TEOS in step II is crucial to the final structures. When TEOS was added earlier (at 3 h instead of 6 h), no obvious bilayered structures were observed for both the invaginated and intact carbon particles (Figure S11). Instead, the structures exhibit single layered mesoporous carbon shells. When TEOS was added at 24 h, only hollow microporous carbon structures with thickness of 15 nm are obtained (Figure S12). These results demonstrate that carefully playing with the polymerization kinetics and elaborately regulating the nucleation process of TEOS and RF precursors in sequence is crucial to obtain bilayered MHCS. To investigate the parameters influencing the invagination of hollow particles, we prepared a series of single layered hollow carbon spheres with controlled wall thicknesses. The wall thickness was controlled from 5 to 16 nm via the increase in reaction time from 6 to 36 h (step I in the scheme). With a thickness as thin as 5 nm, most hollow carbon spheres show invaginated morphology; increasing the thickness to 8 nm leads to a small fraction of invaginated spheres; further increasing the thickness to 13 nm yields only intact spheres (Figure S13). This study demonstrates that the thickness of carbon layer plays a crucial role in controlling the invaginated or intact morphologies of the final products. The distance between the shells was tuned from 7 to 27 nm by increasing the amount of TEOS from 0.5 to 2.5 ml added in step II (Figures S14-17). Notably, when the inter-

layer spacing is enlarged to 27 nm and hydrothermal treatment is applied, an unprecedented structure with the inner layer invaginated while the outer layer remains intact (so called endo-invaginated structure) is obtained (Figure 2c and d, Figure S18 and Movie S2). The ET slide in Figure 2d reveals the cross-sectional crescent and spherical morphology of the inner and outer layers, respectively. The invagination of the inner shell can be ascribed to the formation of a thick and continuous silica layer during step II, which limits the interpenetration of RF and thus decreases the thickness and density of the carbon bridges. It is also noted that shell thickness of the outer sphere is thicker compared to that of the inner invaginated one (Figure 2d), which is attributed to the hydrothermal treatment. With reduced support from bridging between the outer and inner shell, the more flexible inner sphere with thinner walls partially detaches and collapses away from the thicker, intact outer shell, forming the unique endo-invaginated MHCS. Mesoporous hollow nanostructures with high loading capacity are favorable for biomedical applications.36 To evaluate the loading capacity of invaginated and intact MHCS, lysozyme and cyanine dye (Cy3)-labeled oligoDNA (Cy3-oligoDNA) were chosen as model biomolecules. For both invaginated and intact particles, around 75% of saturated adsorption of lysozyme can be achieved within 10 minutes, suggesting fast adsorption kinetics (Figure S19). The maximum adsorption amount for the invaginated particles is ~ 1250 µg mg-1, which is larger than that of intact MHCS (580 µg mg-1) and among the best adsorption performance towards lysozyme compared to previous reports.37, 38 The fast adsorption rate and high adsorption capacity can be attributed to the large entrance size, high surface area, and the hydrophobicity of the invaginated MHCS. The loading capacity of invaginated MHCS toward Cy3-oligoDNA is 252±3 µg mg-1 (Figure S20), which is much higher compared to that of intact MHCS (120±2 µg mg-1), cationic polymer modified large pore mesoporous silica nanoparticles (MSNs) (57 µg mg-1),39 and amine functionalized MSNs with a large pore size of 23 nm (173 µg mg-1).40 The long term colloidal

Figure 4. Confocal microscopy images of HCT-116 cells treated with free Cy3-OligoDNA (100 nM) (first row) and the -1 complex of invaginated MHCS (15 µg ml ) and Cy3OligoDNA (100 nM) (second row). The cytoskeletons and nuclei were stained by Alexa Fluor® 488 phalloidin (green) and DAPI (blue), respectively.



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Figure 5. Hemolysis assay (a) for invaginated and intact MHCS. The digital images (inset) are the supernatants of RBCs suspen-1 -1 sions treated with of invaginated MHCS (80 µg ml ), intact MHCS (80 µg ml ), PBS as a negative control (-) and deionized water as a positive control (+) at room temperature for 2 h. SEM images of RBCs incubated with intact MHCS (b-c) and invaginated MHCS (d-f) at room temperature for 2 h.

stability of invaginated MHCS in biological media is excellent (Figure S21), which is crucial for biomedical applications. The intracellular delivery efficiency of Cy3-oligoDNA using invaginated MHCS as nanocarriers was evaluated in human colon cancer cells (HCT-116). As shown in Figure 4, no Cy3 signals (red color) are observed when the cells are treated with free Cy3-oligoDNA, indicating that the negatively charged biomolecules cannot be taken up by cells. In contrast, the cells treated with the complexes of Cy3-oligoDNA and invaginated MHCS demonstrate strong Cy3 signals within the cells, suggesting that MHCS are efficient nanocarriers for the cellular delivery of Cy3oligoDNA. Both invaginated and intact MHCS show little in vitro cytotoxicity (Figure S22). We further investigated the hemolytic properties of invaginated and intact MHCS with mouse red blood cells (RBCs) for potential intravenous delivery of biomolecules. As displayed in Figure 5a, invaginated MHCS shows a better hemocompatibility compared to intact MHCS at a concentration as high as 80 µg ml-1 (8.2±0.4% vs 12.0±1.2% hemolysis). To explain for the observed difference between two particles in hemolysis, the contact mode of MHCS with RBCs was examined by SEM (Figure 5b-f). Both RBC and intact MHCS have an

isotropic spherical morphology, thus there existed only one contact model (as shown in Figure 5b) for the gradual wrapping of intact MHCS by RBC membrane (Figure 5c). However, for invaginated MHCS with an anisotropic morphology, three typical contact modes were observed through the intact spherical surface (Figure 5d), the edge (Figure 5e) and entire plane of invaginated face (Figure 5f). Particle encapsulation by RBCs mainly depends on the binding energy of particles with RBC membranes and bending energy of RBC membranes.41 The encapsulation occurs when the released binding energy overcomes the energy required to bend RBC membranes. The binding energy relies on the contact area between two particles and thus increases with the contact area. It is postulated that the contact area for the two contact modes shown in Figures 5e (through edge) and f (through the invaginated surface) is less compared to that for the isotropic contact mode (Figures 5b and d), thus invaginated MHCS is harder to be encapsulated by RBCs compared to the intact counterparts and shows better hemocompatibility. Although invaginated hollow structures have been reported before for small anticancer drug delivery,42 there is no report on the comparison of bio-interaction between invaginated and intact morphology. Our work for the first


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time demonstrates the advantage of invaginated morphology over the intact counterparts in hemocompatibility. The unique characteristics including large pore sizes, high loading capacity and excellent hemocompatibility endow the invaginated MHCS a great potential in intravascular delivery of large biomolecules. More exploration on invaginated MHCS is on the way towards various biomedical applications. Conclusions In summary, mesostructured hollow carbon nanoparticles with controllable pore sizes, bi- and triple-layered walls, and adjustable morphologies including invaginated, endo-invaginated and intact spheres, have been synthesized via a surfactant-free sequential heterogeneous nucleation pathway. The invaginated structure shows a high loading capacity of biomolecules compared to the intact structure and efficient intracellular delivery capability of biomolecules. We further demonstrate the advantages of the invaginated morphology over the intact one in hemocompatibility. Our success has paved the way for the designed synthesis of unprecedented mesoporous carbon nanoparticles with potential in diverse biomedical applications. It is envisioned that the SHN pathway can be applied to the self-organization of other polymerizable systems to generate nanostructured interpenetrating composites and eventually new nanomaterials with precisely adjusted structures and wide applications.

ASSOCIATED CONTENT Supporting Information. Other experimental details and characterizations along with additional discussion and supporting data. This material is available free of charge via the Internet at http://pubs.acs.org

AUTHOR INFORMATION Corresponding Author E-mail: [email protected]

Author Contributions The manuscript was written through contributions of all authors.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT The authors acknowledge the financial support from the Australian Research Council, the Queensland Government, the CAS/SAFEA International Partnership Program for Creative Research Teams, the Australian National Fabrication Facility and the Australian Microscopy and Microanalysis Research Facility at the Centre for Microscopy and Microanalysis, The University of Queensland.

REFERENCES 1. Oh, M.; Mirkin, C. A., Chemically tailorable colloidal particles from infinite coordination polymers. Nature 2005, 438, 651-654.

2. Zou, H.; Wu, S. S.; Shen, J., Polymer/silica nanocomposites: Preparation, characterization, properties, and applications. Chem. Rev. 2008, 108, 3893-3957. 3. Li, C. C.; Zeng, H. C., Coordination Chemistry and Antisolvent Strategy to Rare-Earth Solid Solution Colloidal Spheres. J. Am. Chem. Soc. 2012, 134, 19084-19091. 4. Guo, X. H.; Deng, Y. H.; Tu, B.; Zhao, D. Y., Facile Synthesis of Hierarchically Mesoporous Silica Particles with Controllable Cavity in Their Surfaces. Langmuir 2010, 26, 702-708. 5. Stöber, W.; Fink, A.; Bohn, E., Controlled growth of monodisperse silica spheres in the micron size range. J. Colloid Interface Sci. 1968, 26, 62-69. 6. Livage, J.; Lemerle, J., Transition-Metal Oxide Gels and Colloids. Annu. Rev. Mater. Sci. 1982, 12, 103-122. 7. Sun, Y. G.; Xia, Y. N., Shape-controlled synthesis of gold and silver nanoparticles. Science 2002, 298, 2176-2179. 8. Jeong, U.; Wang, Y. L.; Ibisate, M.; Xia, Y. N., Some new developments in the synthesis, functionalization, and utilization of monodisperse colloidal spheres. Adv. Funct. Mater. 2005, 15, 1907-1921. 9. Liu, K.; Nie, Z. H.; Zhao, N. N.; Li, W.; Rubinstein, M.; Kumacheva, E., Step-Growth Polymerization of Inorganic Nanoparticles. Science 2010, 329, 197-200. 10. Pang, X. C.; Zhao, L.; Han, W.; Xin, X. K.; Lin, Z. Q., A general and robust strategy for the synthesis of nearly monodisperse colloidal nanocrystals. Nat. Nanotechnol. 2013, 8, 426-431. 11. Zhao, J. M.; Niu, W. X.; Zhang, L.; Cai, H. R.; Han, M. Y.; Yuan, Y. L.; Majeed, S.; Anjum, S.; Xu, G. B., A Template-Free and Surfactant-Free Method for High-Yield Synthesis of Highly Monodisperse 3-Aminophenol-Formaldehyde Resin and Carbon Nano/Microspheres. Macromolecules 2013, 46, 140-145. 12. Wang, S.; Li, W. C.; Hao, G. P.; Hao, Y.; Sun, Q.; Zhang, X. Q.; Lu, A. H., Temperature-Programmed Precise Control over the Sizes of Carbon Nanospheres Based on Benzoxazine Chemistry. J. Am. Chem. Soc. 2011, 133, 15304-15307. 13. Liu, R.; Mahurin, S. M.; Li, C.; Unocic, R. R.; Idrobo, J. C.; Gao, H. J.; Pennycook, S. J.; Dai, S., Dopamine as a Carbon Source: The Controlled Synthesis of Hollow Carbon Spheres and Yolk-Structured Carbon Nanocomposites. Angew. Chem. Int. Ed. 2011, 50, 6799-6802. 14. Fang, Y.; Gu, D.; Zou, Y.; Wu, Z. X.; Li, F. Y.; Che, R. C.; Deng, Y. H.; Tu, B.; Zhao, D. Y., A Low-Concentration Hydrothermal Synthesis of Biocompatible Ordered Mesoporous Carbon Nanospheres with Tunable and Uniform Size. Angew. Chem. Int. Ed. 2010, 49, 7987-7991. 15. Lu, A. H.; Sun, T.; Li, W. C.; Sun, Q.; Han, F.; Liu, D. H.; Guo, Y., Synthesis of Discrete and Dispersible Hollow Carbon Nanospheres with High Uniformity by Using Confined Nanospace Pyrolysis. Angew. Chem. Int. Ed. 2011, 50, 11765-11768. 16. Liu, Y. L.; Ai, K. L.; Lu, L. H., Polydopamine and Its Derivative Materials: Synthesis and Promising Applications in Energy, Environmental, and Biomedical Fields. Chem. Rev. 2014, 114, 5057-5115. 17. Kim, T. W.; Chung, P. W.; Slowing, I. I.; Tsunoda, M.; Yeung, E. S.; Lin, V. S. Y., Structurally Ordered Mesoporous Carbon Nanoparticles as Transmembrane Delivery Vehicle in Human Cancer Cells. Nano Lett. 2008, 8, 3724-3727. 18. Chen, X. C.; Kierzek, K.; Jiang, Z. W.; Chen, H. M.; Tang, T.; Wojtoniszak, M.; Kalenczuk, R. J.; Chu, P. K.; Borowiak-Palen, E., Synthesis, Growth Mechanism, and Electrochemical Properties of Hollow Mesoporous Carbon Spheres with Controlled Diameter. J. Phys. Chem. C 2011, 115, 17717-17724. 19. Li, N.; Zhang, Q.; Liu, J.; Joo, J.; Lee, A.; Gan, Y.; Yin, Y., Sol-gel coating of inorganic nanostructures with resorcinolformaldehyde resin. Chem. Commun. 2013, 49, 5135-5137. 20. Guan, B. Y.; Wang, X.; Xiao, Y.; Liu, Y. L.; Huo, Q. S., A versatile cooperative template-directed coating method to con-



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struct uniform microporous carbon shells for multifunctional core-shell nanocomposites. Nanoscale 2013, 5, 2469-2475. 21. Liu, Y. D.; Goebl, J.; Yin, Y. D., Templated synthesis of nanostructured materials. Chem. Soc. Rev. 2013, 42, 2610-2653. 22. Ma, T. Y.; Liu, L.; Yuan, Z. Y., Direct synthesis of ordered mesoporous carbons. Chem. Soc. Rev. 2013, 42, 3977-4003. 23. Zhang, P.; Qiao, Z.-A.; Dai, S., Recent advances in carbon nanospheres: synthetic routes and applications. Chem. Commun. 2015, 10.1039/c5cc01759a. 24. Gu, D.; Bongard, H.; Deng, Y. H.; Feng, D.; Wu, Z. X.; Fang, Y.; Mao, J. J.; Tu, B.; Schuth, F.; Zhao, D. Y., An Aqueous Emulsion Route to Synthesize Mesoporous Carbon Vesicles and Their Nanocomposites. Adv. Mater. 2010, 22, 833. 25. Liu, J.; Qiao, S. Z.; Liu, H.; Chen, J.; Orpe, A.; Zhao, D. Y.; Lu, G. Q., Extension of The Stöber Method to the Preparation of Monodisperse Resorcinol-Formaldehyde Resin Polymer and Carbon Spheres. Angew. Chem. Int. Ed. 2011, 50, 5947-5951. 26. Zhang, X. H.; Li, Y. A.; Cao, C. B., Facile one-pot synthesis of mesoporous hierarchically structured silica/carbon nanomaterials. J. Mater. Chem. 2012, 22, 13918-13921. 27. Qiao, Z. A.; Guo, B. K.; Binder, A. J.; Chen, J. H.; Veith, G. M.; Dai, S., Controlled Synthesis of Mesoporous Carbon Nanostructures via a "Silica-Assisted" Strategy. Nano Lett. 2013, 13, 207-212. 28. Fang, X. L.; Zang, J.; Wang, X. L.; Zheng, M. S.; Zheng, N. F., A multiple coating route to hollow carbon spheres with foam-like shells and their applications in supercapacitor and confined catalysis. J. Mater. Chem. A 2014, 2, 6191-6197. 29. Fuertes, A. B.; Valle-Vigon, P.; Sevilla, M., One-step synthesis of silica@resorcinol-formaldehyde spheres and their application for the fabrication of polymer and carbon capsules. Chem. Commun. 2012, 48, 6124-6126. 30. Friedrich, H.; de Jongh, P. E.; Verkleij, A. J.; de Jong, K. P., Electron Tomography for Heterogeneous Catalysts and Related Nanostructured Materials. Chem. Rev. 2009, 109, 1613-1629. 31. Yang, J.; Zhang, H.; Yu, M.; Emmanuelawati, I.; Zou, J.; Yuan, Z.; Yu, C., High-Content, Well-Dispersed γ-Fe2O3 Nanoparticles Encapsulated in Macroporous Silica with Superior Arsenic Removal Performance. Adv. Funct. Mater. 2014, 24, 13541363. 32. Kremer, J. R.; Mastronarde, D. N.; McIntosh, J. R., Computer visualization of three-dimensional image data using IMOD. J. Struct. Biol. 1996, 116, 71-76. 33. Kristensen, E. M. E.; Nederberg, F.; Rensmo, H.; Bowden, T.; Hilborn, J.; Siegbahn, H., Photoelectron spectroscopy studies of the functionalization of a silicon surface with a phosphorylcholine-terminated polymer grafted onto (3aminopropyl)trimethoxysilane. Langmuir 2006, 22, 9651-9657. 34. Bogush, G. H.; Zukoski, C. F., Studies of the Kinetics of the Precipitation of Uniform Silica Particles through the Hydrolysis and Condensation of Silicon Alkoxides. J. Colloid Interface Sci. 1991, 142, 1-18. 35. Sear, R. P., Nucleation: theory and applications to protein solutions and colloidal suspensions. J. Phys.: Condens. Mat. 2007, 19, 03310. 36. Chen, Y.; Chen, H. R.; Shi, J. L., Construction of Homogenous/Heterogeneous Hollow Mesoporous Silica Nanostructures by Silica-Etching Chemistry: Principles, Synthesis, and Applications. Acc. Chem. Res. 2014, 47, 125-137. 37. Ding, Y.; Yin, G. F.; Liao, X. M.; Huang, Z. B.; Chen, X. C.; Yao, Y. D.; Li, J., A convenient route to synthesize SBA-15 rods with tunable pore length for lysozyme adsorption. Microporous Mesoporous Mater. 2013, 170, 45-51. 38. Lan, F.; Hu, H.; Jiang, W.; Liu, K. X.; Zeng, X. B.; Wu, Y.; Gu, Z. W., Synthesis of superparamagnetic Fe3O4/PMMA/SiO2 nanorattles with periodic mesoporous shell for lysozyme adsorption. Nanoscale 2012, 4, 2264-2267.

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39. Hartono, S. B.; Gu, W. Y.; Kleitz, F.; Liu, J.; He, L. Z.; Middelberg, A. P. J.; Yu, C. Z.; Lu, G. Q.; Qiao, S. Z., Poly-L-lysine Functionalized Large Pore Cubic Mesostructured Silica Nanoparticles as Biocompatible Carriers for Gene Delivery. ACS Nano 2012, 6, 2104-2117. 40. Na, H. K.; Kim, M. H.; Park, K.; Ryoo, S. R.; Lee, K. E.; Jeon, H.; Ryoo, R.; Hyeon, C.; Min, D. H., Efficient Functional Delivery of siRNA using Mesoporous Silica Nanoparticles with Ultralarge Pores. Small 2012, 8, 1752-1761. 41. Zhao, Y. N.; Sun, X. X.; Zhang, G. N.; Trewyn, B. G.; Slowing, I. I.; Lin, V. S. Y., Interaction of Mesoporous Silica Nanoparticles with Human Red Blood Cell Membranes: Size and Surface Effects. ACS Nano 2011, 5, 1366-1375. 42. Chen, Y.; Xu, P. F.; Wu, M. Y.; Meng, Q. S.; Chen, H. R.; Shu, Z.; Wang, J.; Zhang, L. X.; Li, Y. P.; Shi, J. L., Colloidal RBCShaped, Hydrophilic, and Hollow Mesoporous Carbon Nanocapsules for Highly Efficient Biomedical Engineering. Adv. Mater. 2014, 26, 4294-4301.


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Self-organized Mesostructured Hollow Carbon Nanoparticles via a Surfactant-free Sequential Heterogeneous Nucleation Pathway


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Self-Organized Mesostructured Hollow Carbon Nanoparticles via a

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