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Dec 6, 2013 - ABSTRACT: A novel high level oriented assembly of zinc hydroxide carbonate nanocrystals in chitosan was fabricated via a hydrothermal ...
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Nanocrystal−Organic Hybrid Antifungal Agent: High Level Oriented Assembly of Zinc Hydroxide Carbonate Nanocrystals in Chitosan Shisen Song,† Bo You,*,† Yingchun Zhu,*,‡ Yandan Lin,§ Yin Wu,∥ and Xiaochun Ge∥ †

Department of Materials Science and the Advanced Coatings Research Center of the China Educational Ministry, Fudan University, 220 Handan Road, Shanghai 200433, PR China ‡ Key Laboratory of Inorganic Coating Materials, Shanghai Institute of Ceramics, Chinese Academy of Science, Shanghai 200050, PR China § School of Information Science and Engineering, Fudan University, Shanghai 200433, PR China ∥ The School of Life Sciences, Fudan University, Shanghai 200433, PR China S Supporting Information *

ABSTRACT: A novel high level oriented assembly of zinc hydroxide carbonate nanocrystals in chitosan was fabricated via a hydrothermal process. Hydrated chitosan acts as a template supporting and structure-directing reagents for the formation and assembly of zinc hydroxide carbonate nanocrystals. Zinc hydroxide carbonate nanocrystals were heterogeneously nucleated at the interface of hydrated chitosan crystallization regions through hydrogen bonding, lattice geometrical matching, and stereochemical matching interactions with ordered chitosan chains. The products obtained at different reaction stages were characterized with SEM, TEM, XRD, XPS, and TGA. The antifungal activity assays of CZHC, zinc hydroxide carbonate, chitosan, chitosan simply blended with zinc hydroxide carbonate (1:1), and nano zinc oxide were investigated. The study exhibited that the CZHC nanocrystals showed an antifungal activity against Cotton Verticillium, Rhizopus, and Mucorales and showed a better antifungal activity than other test samples against Rhizopus.

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INTRODUCTION Nanocrystal−organic hybrid materials are masterpieces of natural organisms that control the nucleation and growth of nanocrystals and assemble them into ordered superstructures in an organic matrix via a biomineralization process.1−3 Such biological phenomena stimulated many attempts to study and mimic the biomineralization process.4−6 Inspired from the recent achievements in biomimicry, we use chitosan as an organic matrix to prepare zinc hydroxide carbonate nanocrystal assembly. Zinc hydroxide carbonate is a versatile chemical material which is widely used in medicine and industries.7−12 It has attracted considerable interest due to its antimicrobial and antifungal activity,13 but inorganic nanoparticles are prone to adhering to each other and form agglomerations, making it hard to adhere and interact with the microorganism. Chitosan is one of the important biomacromolecules, which not only shows interests in biomineralization14−17 but also provide a variety of biomedical applications such as wound healing, dermatology, anti-itching, and drug delivery, etc.17 As a biomacromolecule with hydroxyl and amino groups, chitosan is hydrophilic, biocompatible, and easily interacts with the microorganism.18 With anticipation to combine the functions of zinc hydroxide carbonate and chitosan, herein we studied the formation process of zinc hydroxide carbonate nanocrystals in a chitosan matrix and their antifungal activity. © 2013 American Chemical Society

Nanotechnology opens a new innovation to iatrology including numerous diagnostics and medicines owing to the unusual physicochemical properties of nanomaterials.19−25 Interesting antibacterial activity has been found in the many nanoparticles, which shines light on developing new antimicrobial agents.26−30 Microorganism infection via bacteria, fungi, and viruses causes disastrous effects on mammalians and plants.31−37 Especially, fungi are harder nuts to crack due to their special structures than other microorganisms.36,38−40 Antifungal agents are demanded because conventional antibiotics are to too expensive to inhibit fungi of plants, and many of them are not effective to fungal diseases.37,41−43 Although there are many studies on iatrology based on nanotechnology,23,44,45 only a few are reported on antifungal agents including silver and zinc oxide nanoparticles27,46,47 In this work, we report an oriented assembly of zinc hydroxide carbonate nanocrystals with size less than 10 nm in chitosan matrix. By controlling the reaction conditions, zinc hydroxide carbonate with high specific surface area are uniformly and orientedly dispersed in chitosan matrix, leading to a better antifungal effect against Rhizopus, Cotton Verticillium, and Mucorales. Received: July 11, 2013 Revised: November 10, 2013 Published: December 6, 2013 38

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Article

EXPERIMENTAL SECTION

Materials. Zinc acetate (Zn(AC)2·2H2O), urea (CO(NH2)2), acetic acid (HAC, 99.5%), chitosan (degree of acetylation (DA) ≤ 10%), zinc hydroxide carbonate, nano zinc oxide, and sodium hydroxide (NaOH) were purchased from Guoyao Chemical Reagent Co. (China). All chemicals are analytical-grade regents. They were used without further purification. Synthesis of Chitosan/Zinc Hydroxide Carbonate. In a typical process, a total of 0.20 g of chitosan was dissolved in 30 mL of deionized water containing 0.1 mL of acetic acid to form a transparent solution. Then 2.5 g of zinc acetate (Zn(AC)2·2H2O) was dissolved in 30 mL of deionized water and 4 g of urea was introduced under vigorous stirring to form a homogeneous solution, and the mixed solution was added into 30 mL of the above chitosan solution. Then 1 mol/L sodium hydroxide solution was used to adjust the pH = 7.5 in the above solution, and a white precipation was obtained. Finally the gel was transferred into a Teflon-lined autoclave of 100 mL capacity. The autoclave was heated to 90 °C for 6, 12, or 24 h. After being cooled to room temperature, the white powders were collected and washed several times with distilled water and ethanol to remove the impurities and then dried at ambient temperature for 24 h. Characterization. SEM Observation. Scanning electron microscopy (SEM) combined with energy dispersive X-ray (EDX) analysis was performed on a Philips XL 30 field emission microscope. All samples were coated with gold by sputtering prior to observation. TEM Observation. Transmission electron microscopy (TEM), select-area electron diffraction (SAED), and high-resolution transmission electron microscopy (HRTEM) were obtained on a JEM-2010 microscope with an accelerating voltage of 200 kV. XRD Analysis. Powder X-ray diffraction (XRD) was recorded on a D/max 2550 V diffractometer with Cu Kα radiation (λ) 1.542 Å). DTA-TG Analysis. Differential thermal analysis−thermogravimetry (DTA-TG) was performed on NETZSCH STA 429C. The heating rate used was 10 °C/min under an air flow of 20 mL/min from 30 to 800 °C. XPS survey. XPS detection was performed by a Perkin-Elmer PHI 5000 C ESCA system using Al Kα radiation (1486.6 eV) at a power of 250 W. The pass energy was set at 93.9 eV, and the binding energy (BE) of 285.0 eV for the C 1s of aliphatic carbons was taken as the reference energy. The XPS analysis was done at room temperature at 90° electron takeoff angle. Antifungal Activity Assay. Four types of plant fungi were used for antifungal activity assay. They are Cotton Verticillium, Coniothyrium diplodiella (Speg.), Rhizopus, and Mucorales. Antifungal activity was measured by microspectrophotometry. Routinely, tests were performed with 20 μL of sample with different amounts of CZHC powders and 80 μL of a suspension of fungal spores (2 × 104 spores mL−1) in potato dextrose broth liquid media; pH of culture solution is 6.4. All samples were tested in 96-well plates and incubated at 25 °C. The control microcultures have the same ingredients as sample microcultures but excluding the CZHC powders. After incubated for 48 h, the absorption of each well was calculated by microspectrophotometry at the wavelength of 405 nm, as shown as Supporting Information Table S1. The antifungal activity assay of zinc hydroxide carbonate, chitosan, chitosan/zinc hydroxide carbonate (chitosan simply blended with zinc hydroxide carbonate, the mass ratio of 1 to1), and nano zinc oxide were also tested as comparison with CZHC, as shown in Supporting Information Table S2.

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Figure 1. XRD patterns of the samples obtained with different hydrothermal treatment time at 90 °C, (a) 0 h, pH = 7.5, (b) 6 h, pH = 7.8, (c) 12 h, pH = 8.5, (d) 18 h, pH = 8.8, (e) 24 h, pH = 9.0.

and 24 h, the pH values of solutions were further increased to 8.5, 8.8, and 9.0, and most of the amorphous phase samples turned into crystalline Zn5(CO3)2(OH)6 in this process. In addition, the relative diffraction density of (200) planes of Zn5(CO3)2(OH)6 crystals increased with increasing reaction time and became the strongest one after 24 h, meaning an evolution of the morphology and orientation of Zn5(CO3)2(OH)6 nanocrystals. The morphologies of the products at different stages were revealed by scanning electron microscopy (SEM) as shown in Figure 2. We can see the typical morphology of the initial amorphous precipitation (Figure 2a). Such initial solid precipitation got mesoporous, showing facet-like surfaces after 6 h reaction (Figure 2b), which has a crystalline structure of Zn5(CO3)2(OH)6 (Figure 1b). The products got delaminated when a large quantity of Zn5(CO3)2(OH)6 was formed after 12 h (Figures 2c and 1c). When the reaction was carried out for 18

RESULTS AND DISCUSSION

The X-ray diffraction (XRD) diffraction patterns of products obtained at different reaction stages were shown in Figure 1. An amorphous phase samples were obtained when the zinc acetate and urea were introduced in the chitosan gel with the pH value of solution adjusted to 7.5 (Figure 1a). White products with crystalline Zn5(CO3)2(OH)6 (JCPDS 19-1458) were prepared after 6 h reaction with the pH value of the solution increased to 7.8 (Figure 1b). When reactions were carried out for 12, 18,

Figure 2. SEM images of samples obtained with different hydrothermal treatment time at 90 °C, (a) 0 h, (b) 6 h, (c) 12 h, (d) 18 h, (e) 24 h, (f) high magnification of (e). 39

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Figure 3. XPS spectra (a) and TGA analysis (b) of CZHC sample and pure chitosan. Inset in (a) XPS spectra of C element.

h, most of the precipitations were transformed into sheet-like structures (Figure 2d). Finally, the products had been obtained as sheets with a uniform thickness about 20 nm and broad in micrometer scale after 24 h reaction (Figure 2e). The sheets had surfaces with rough textures as revealed by the high magnification SEM image (Figure 2f). To further investigate the composition of the sheet products obtained after 24 h reaction, the product and pure chitosan were analyzed by the X-ray photoelectron spectroscopy (XPS) and thermogravimetry analysis (TGA) as shown in Figure 3. The peaks at binding energies of 95, 145, 285, 428, 535, and 1052 eV, corresponding to Zn 3p3, Zn3s, C1s, N1s, O1s and Zn2p3, respectively, can be observed (Figure 3a). The peaks at binding energies of 285, 428, and 535 eV correspond to C1s, N1s, and O1s of chitosan, respectively (Figure 3a), suggesting that the sample is composed of both chitosan and zinc compound. By analyzing the C1s band profile of the sheet products (Inset in Figure 3a), the broad line was resolved into two components center centered at BE = 285 and 289 eV, which was ascribed to chitosan and Zn5(CO3)2(OH)6 species, respectively.48,49 The area ratio of the peak at 285 eV and that at 289 eV is 3.13. The atomic ratio of CZHC sample is calculated from XPS spectra, and C/O/Zn atomic ratio is 42.07:42.06:12.68%. The zinc hydroxide carbonate (Zn5(CO3)2(OH)6, M = 549) contents in the CZHC sample is estimated to be about 57% from XPS spectra. The TGA analyses of the products show a tremendous weight loss between 150 and 330 °C owing to the decomposition of zinc hydroxide carbonate and chitosan. The weight loss between 330 and 450 °C was contributed to the further decomposition of zinc carbonate. Total weight loss at 800 °C was 45% for the zinc hydroxide carbonate composite, while the theoretical weight loss of Zn5(CO3)2(OH)6 in nitrog en atm osp here was 26%. The content of Zn5(CO3)2(OH)6 in the composite was estimated to be about 44%, which was smaller than that analyzed with XPS spectra. Foregoing results show that the products are composed of Zn5(CO3)2(OH)6 and chitosan, and the Zn5(CO3)2(OH)6 is more likely to assemble in the surface of the sheet product . The structure of the chitosan/zinc hydroxide carbonate (CZHC) products is further analyzed by transmission electron microscopy (TEM) as shown in Figure 4a. The selected area electron diffraction (SAED) analysis revealed that the sheets are crystalline (inset of Figure 4a). The diffraction patterns can be indexed as those of the (020) and (001) planes of the zinc hydroxide carbonate, which is consistent with the XRD analysis (Figure 1). We find that the chitosan/zinc hydroxide carbonate products can be fragmented and dispersed in water under sonication. The broken pieces of the CZHC sample are shown in Figure 4b. HRTEM images show that the sheet-like

Figure 4. (a) TEM images of a sheet-like chitosan/zinc hydroxide carbonate (CZHC) products. The inset shows the selected area SAED pattern (b, c, d) high-resolution TEM (HRTEM) lattice image of the zinc hydroxide carbonate nanocrystals.

structures are composed of zinc hydroxide carbonate nanocrystals which are orientedly assembled in chitosan as depicted in Figure 4c,d. Foregoing investigation shows that the sheet products obtained after 24 h reaction contain ca. 44 wt % Zn5(CO3)2(OH)6 and ca. 56 wt % chitosan; nanocrystals of Zn5(CO3)2(OH)6 are uniformly and orientedly dispersed in chitosan matrix. The formation process of nanostructured chitosan/zinc hydroxide carbonate probably involves a three-stage process. First, when urea and zinc acetate were added into chitosan aqueous solution and the pH value of solution was adjusted to 7.5, a white precipitate was obtained because chitosan sol changed into gel. Second, when urea was decomposed into carbon dioxide (CO2) and ammonia (NH3·H2O) (eq 1), the evolved carbon dioxide (CO2) reacted with water forming carbonate ion (CO32−) (eq 2). The pH value of the system was increased in this process, and then zinc cation (Zn2+) reacted with carbonate ion (CO32−) and hydroxyl (OH−), forming zinc hydroxide carbonate nanocrystals (Zn5(CO3)2(OH)6) in a chitosan matrix (eq 3). Zinc hydroxide carbonate (Zn5(CO3)2(OH)6) nanocrystals was heterogeneously loaded on the chitosan macromolecules chain. In the formation process of the assembly of zinc hydroxide carbonate nanocrystals, chitosan acted as template supporting and structuredirecting reagents. The FTIR spectrum of the samples obtained at different state shown in Figure 5 further confirmed the formation process. A broad absorption band coming from −OH groups appears at around 3434 cm−1, and the adsorption peaks of chitosan at 40

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matches with each other in (100) planes. The hydroxyl groups in (100) planes of hydrated chitosan attract carbonate via hydrogen bonding interactions (Figure 6b), which is confirmed from the FTIR spectrum (Figure 5) and then further bounded to zinc ions via electrostatic interactions as the nucleation center of zinc hydroxide carbonate (Figure 6c). Zinc ions further attracts hydroxyl ions and carbonate ions, and zinc hydroxide carbonate crystals are selectively nucleated due to the lattice geometrical matching with the unit cell of hydrated chitosan (Figure 6d). The trigonal planar carbonate ions interact with chitosan chains in vertical mode, as shown in Figure 6b. The stereochemical arrangement of the carbonate ions in conjunction with zinc binding generates a two-layer subunit cell motif of Zn5(CO3)2(OH)6 . Above analyses reveal that hydrated chitosan crystallization regions selectively control the formation of Zn5(CO3)2(OH)6 crystals via hydrogen bonding, lattice geometrical, and stereochemical matching interactions. Cotton Verticillium, Coniothyrium diplodiella (Speg.), Rhizopus, and Mucorales were used for antifungal activity assay. The optical images of fungicidal activities of CZHC against the different fungi with concentrations of 1.0, 10, and 100 mg/L are given in Figure 7. It was shown that there were fungicidal activities of CZHC against three fungi tested (Cotton Verticillium, Rhizopus, and Mucorales) while it had almost no activity against Coniothyrium diplodiella even if the concentration of CZHC is increased from 1 to 100 mg/L. Fungicidal activities of CZHC against three fungi tested (Cotton Verticillium, Rhizopus, and Mucorales) increased with increasing the concentration of CZHC (see Supporting Information Table S1). Among these four fungi, Cotton Verticillium were more sensitive to the CZHC nanocrystals. SEM image of Mucorales incubated in the culture medium containing CZHC for 48 h was shown in Figure 8a,b. It could be seen that the CZHC were disassambled and penetrated the fungi membrane of Mucorales as shown in Figure 8b. CZHC nanoparticles in Mucorales were also observed in TEM image (Figure 8c) and corresponding EDX spectrum (Figure 8d). Generally, the antifungal mechanism of various antibiotics can be divided into three categories: (1) affecting fungal sterols,58 (2) affecting the fungal cell wall and leading to rupture of the membrane,59 and (3) inhibiting nucleic acids.60 It is proposed that the chitosan/zinc hydroxide carbonate nanofungicide may cause denaturation of functional membrane proteins, thus interrupt the normal activities of plasma membranes. CZHC is easily hydrolyzed in aqueous solution forming hydroxyl groups (OH−), which react with oxygen (O2) in the culture solution, producing reactive oxygen species (ROS) and thus cause the destruction of the fungal membrane. Meanwhile, positively charged chitosan and Zn2+ interact with negatively charged fungal cell membranes, causing inhibition of enzyme activities. Further investigations concerning the exact mechanism of the antifungal action of CZHC is under way. The antifungal activity assay of zinc hydroxide carbonate, chitosan, chitosan/zinc hydroxide carbonate (1:1), and nano zinc oxide were also tested as comparison with CZHC. The optical images of fungicidal activities of different samples against Rhizopus with concentrations of 1.0, 10, and 100 mg/L are given in Figure 9. It was shown that conventional zinc compounds such as zinc hydroxide carbonate (Figure 9a) and nano zinc oxide (Figure 9d) had almost no activity against Rhizopus even if the concentration was increased from 1 to 100 mg/L, and chitosan (Figure 9b) and chitosan/zinc hydroxide

Figure 5. FTIR spectra of (a) chitosan sol, (b) chitosan gel, (c) chitosan/zinc hydroxide carbonate (CZHC).

1151 cm−1 (asymmetric stretching of −C−O−C− bridge) and 1034 cm−1 (C−N bond) also appear in the FTIR spectrum of chitosan sol (Figure 5a).50 After chitosan sol changed into gel (Figure 5b), typical absorption bands of NH2 group can be observed: the absorption bands at 3347 and 3251 cm−1 can be assigned to stretching vibration of N−H, and the band at 1626 cm−1 belong to the bending deformation modes of N−H. It is well-known that −NH2 and −OH can form strong H-bonding with −CO or −C-O−.50,51 Therefore, chitosan chains can adsorb carbonate ions at their interface through the hydrogen bonding interactions. The free carbonate ion has the D3h symmetry and exhibits four normal vibration modes, ν1 (1100−1000 cm−1), ν2 (800−650 cm−1), ν3 (1500−1400 cm−1), and ν4 (1000−800 cm−1).52,53 The absorption bands corresponding to the CO32− group can be observed, which indicates that the hydroxyl groups of hydrated chitosan attract carbonate in this process: the absorption band at 1464 cm−1 can be assigned to the asymmetric stretching modes (ν3) and the band at 789 cm−1 should be attributed to the out-of-plane deformation mode (ν2). Typical peaks of chitosan as is seen in Figure 5b are also found in the FTIR spectrum of the final product (Figure 5c), and the absorption bands corresponding to the CO32− group (1420 cm−1, 791 cm−1) and the “Zn−OH” bonding (869 cm−1)54 are also found, which further confirms the composition of the sheet products. NH 2CONH 2 + H 2O → NH4 + + OH− + CO2

(1)

CO2 + 2OH− → CO32 − + H 2O

(2)

5Zn 2 + + 2CO32 − + 6OH− → Zn5(CO3)2 (OH)6

(3)

The schematic illustration of the chitosan/zinc hydroxide carbonate nanocrystal self-assembly is suggested in Figure 6. It is reported that chitosan forms precipitate with orderly aggregated chains in alkaline solutions.55 As is known, hydrated chitosan crystallization regions are in an orthorhombic unit cell with dimensions a1 = 8.95, b1 = 16.97, c1 (ber axis) = 10.34 Å and a space group P212121 in the dispersing state of chitosan precipitates,56 and Zn5(CO3)2(OH)6 is monoclinic with a2 = 13.62, b2 = 6.30, c2 = 5.42 Å, β = 95.5°, and a space group C2/ m.57 From that, we can find that b1 ≈ 3b2, c1 ≈ 2c2, which indicate that the lattice of chitosan and Zn5(CO3)2(OH)6 41

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Figure 6. Schematic illustration of the chitosan/zinc hydroxide carbonate nanocrystal self-assembly process. (a) Schematic graph of chitosan chains, (b) hydroxyl groups of chitosan chains binding with carbonate ions through hydrogen bonding, (c) carbonate ions bounded by chitosan chains attracting calcium ions by electrostatic forces, and (d) stereochemical arrangement of carbonate ions in conjunction with Zn2+ guiding the formation of zinc hydroxide carbonate.

carbonate (Figure 9c) only showed limited fungicidal activities, while CZHC (Figure 9e) showed a better fungicidal activity than others (see Supporting Information Table S2). This further confirms, by forming the hybrid structure, the overall

antifungal properties have been improved, which cannot be achieved by simply blending. The morphology of zinc hydroxide carbonate and nano zinc oxide were shown in Supporting Information Figure S1. It was shown that zinc 42

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Figure 9. Optic images of fungicidal activities of (a) zinc hydroxide carbonate, (b) chitosan, (c) chitosan/zinc hydroxide carbonate (1:1), (d) nano zinc oxide, and (e) CZHC against Rhizopus. Figure 7. Optic images of fungicidal activities of CZHC against the different fungi. (a) Rhizopus, (b) Mucorales, (c) Cotton Verticillium, (d) Coniothyrium diplodiella.

to its larger specific surface area. As the result, the CZHC composite shows a better fungicidal activity than its components.

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hydroxide carbonate and nano zinc oxide are prone to adhering to each other and form agglomerations, making it hard to adhere and interact with the microorganism. As a result, chitosan, zinc compounds, and their mixture show poor fungicidal activities. On the other hand, CZHC show sheetlike morphology with a thickness of 20 nm and broad in micrometer scale (Figure 2e); Zn5(CO3)2(OH)6 are uniformly and orientedly dispersed in chitosan matrix, forming nanocrystals with size less than 10 nm (Figure 4c). The chitosan matrix makes the CZHC a good interaction with the microorganism due to its hydrophilicity and biocompatibility, while the Zn5(CO3)2(OH)6 nanocrystal shows a better fungicidal activity than aggregated nano zinc compounds due

CONCLUSION In summary, we have described a novel biomolecule−inorganic hybrid material CZHC, a high level oriented assembly of zinc hydroxide carbonate nanocrystals in chitosan. The hybrid materials contains ca. 44 wt % Zn5(CO3)2(OH)6 and ca. 56 wt % chitosan, showing sheetlike morphology with a thickness of 20 nm and broad in micrometer scale; nanocrystals of Zn5(CO3)2(OH)6 are uniformly and orientedly dispersed in the chitosan matrix. Hydrated chitosan acts as a template supporting and structure-directing reagents for the formation and assembly of zinc hydroxide carbonate nanocrystals. Zinc

Figure 8. SEM image (a,b), TEM image (c), and corresponding EDX spectrum of (c) of Mucorales after incubating in the culture medium containing CZHC for 48 h. 43

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hydroxide carbonate nanocrystals were heterogeneously nucleated at the interface of hydrated chitosan crystallization regions through hydrogen bonding, lattice geometrical matching, and stereochemical matching interactions with ordered chitosan chains. The antifungal activity assays exhibited that the CZHC nanocrystals showed an antifungal activity against Cotton Verticillium, Rhizopus, and Mucorales. These findings may be particularly useful for the preparation of nanocrystal−organic hybrid superstructures and antifungal applications.

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ASSOCIATED CONTENT

S Supporting Information *

Results of antifungal activity assay of CZHC against different fungi, results of antifungal activity assay of different samples against Rhizopus, SEM image of nano zinc hydroxide carbonate and nano zinc oxide. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (B.Y.). *E-mail: [email protected] (Y.C.Z.) Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support of this research from the Science and Technology of Shanghai (10DZ1141102, 11XD1405600), the Nation Natural Science Foundation of China (51072217, 51232007, 51132009), High-Level Talents Introduction Project in Jiangsu Province, the Shanghai Leading Academic Discipline Project (B113), and the Key Laboratory of Inorganic Coating Materials, Chinese Academy of Sciences project is appreciated.

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